Skip to main content
Persoonia : Molecular Phylogeny and Evolution of Fungi logoLink to Persoonia : Molecular Phylogeny and Evolution of Fungi
. 2021 Jan 13;47:106–135. doi: 10.3767/persoonia.2021.47.03

Species of Botryosphaeriaceae associated with citrus branch diseases in China

XE Xiao 1,2, W Wang 1,2, PW Crous 3,4, HK Wang 1, C Jiao 1, F Huang 5, ZX Pu 6, ZR Zhu 2, HY Li 1,2, *
PMCID: PMC10486630  PMID: 37693792

Abstract

Citrus is an important and widely cultivated fruit crop in South China. Although the species of fungal diseases of leaves and fruits have been extensively studied, the causal organisms of branch diseases remain poorly known in China. Species of Botryosphaeriaceae are known as important fungal pathogens causing branch diseases on citrus in the USA and Europe. To determine the diversity of Botryosphaeriaceae species associated with citrus branch diseases in China, surveys were conducted in the major citrus-producing areas from 2017 to 2020. Diseased tissues were collected from twigs, branches and trunks with a range of symptoms including cankers, cracking, dieback and gummosis. Based on morphological characteristics and phylogenetic comparison of the DNA sequences of the internal transcribed spacer region (ITS), the translation elongation factor 1-alpha gene (tef1), the β-tubulin gene (tub2) and the DNA-directed RNA polymerase II second largest subunit (rpb2), 111 isolates from nine provinces were identified as 18 species of Botryosphaeriaceae, including Botryosphaeria dothidea, B. fabicerciana, Diplodia seriata, Dothiorella alpina, Do. plurivora, Lasiodiplodia citricola, L. iraniensis, L. microconidia, L. pseudotheobromae, L. theobromae, Neodeightonia subglobosa, Neofusicoccum parvum, and six previously undescribed species, namely Do. citrimurcotticola, L. guilinensis, L. huangyanensis, L. linhaiensis, L. ponkanicola and Sphaeropsis linhaiensis spp. nov. Botryosphaeria dothidea (28.8 %) was the most abundant species, followed by L. pseudotheobromae (23.4 %), which was the most widely distributed species on citrus, occurring in six of the nine provinces sampled. Pathogenicity tests indicated that all 18 species of Botryosphaeriaceae obtained from diseased citrus tissues in this study were pathogenic to the tested Citrus reticulata shoots in vitro, while not all species are pathogenic to the tested Cocktail grapefruit (C. paradisi × C. reticulata) shoots in vivo. In addition, Lasiodiplodia was the most aggressive genus both in vitro and in vivo. This is the first study to identify Botryosphaeriaceae species related to citrus branch diseases in China and the results provide a theoretical basis for the implementation of prevention and control measures.

Citation: Xiao XE, Wang W, Crous PW, et al. 2021. Species of Botryosphaeriaceae associated with citrus branch diseases in China. Persoonia 47: 106–135. https://doi.org/10.3767/persoonia.2021.47.03.

Keywords: Botryosphaeria cankers, distribution, new taxa, pathogenicity, systematics

INTRODUCTION

The Botryosphaeriaceae was established by Theissen & Sydow (1918). The taxonomic status of Botryosphaeriaceae has been heavily debated and somewhat controversial until Schoch et al. (2006) proposed the Botryosphaeriales as a new order to accommodate the family (see Phillips et al. 2013). Presently, the Botryosphaeriaceae contains 23 genera and over 100 species that have been confirmed based on their DNA sequence data (Slippers et al. 2017, Yang et al. 2017, Zhang et al. 2021).

Species of Botryosphaeriaceae have a broad host range and cosmopolitan distribution (Slippers & Wingfield 2007, Phillips et al. 2013). Many species are important plant pathogens, especially for woody plant genera such as Citrus, causing bark rot, branch canker, gummosis, shoot blight, dieback and fruit rot, and even death of whole plants when conditions are conducive to disease development (Slippers & Wingfield 2007, Úrbez-Torres 2011). Citrus is one of the most important fruit crops globally. Citrus diseases caused by species in the Botryosphaeriaceae have been reported since the early 1900s when Fawcett & Burger (1911) isolated a Diplodia sp. from orange trees with gummosis, and from rotten grapefruits and oranges in Florida. The fungal agent was then considered to be Diplodia natalensis, which was regarded as the pathogen responsible for decay and gummosis in lemons and other citrus fruits in the USA and South Africa (Fawcett & Burger 1911, Adesemoye et al. 2014). Subsequent taxonomic revisions showed that D. natalensis represents as synonym of Lasiodiplodia theobromae (Alves et al. 2004). Further studies indicated that Diplodia stem-end rot caused by L. theobromae is one of the most important postharvest decays in warm, humid tropical and subtropical citrus-producing areas (Brown & Eckert 2000, Ismail & Zhang 2004, Zhang 2014). Several other species of Botryosphaeriaceae have subsequently been isolated from citrus with cankers, dieback, gummosis and fruit rot symptoms, including species of Botryosphaeria (Smith 1934, Adesemoye et al. 2011), Diplodia (Adesemoye et al. 2014, Berraf-Tebbal et al. 2020), Dothiorella (Adesemoye & Eskalen 2011, Abdollahzadeh et al. 2014, Berraf-Tebbal et al. 2020), Lasiodiplodia (Alves et al. 2008, Abdollahzadeh et al. 2010, Adesemoye et al. 2014, Linaldeddu et al. 2015, Coutinho et al. 2017, Guajardo et al. 2018, Bautista-Cruz et al. 2019, Berraf-Tebbal et al. 2020), Macrophomina (Azadeh et al. 2018), Neofusicoccum (Adesemoye & Eskalen 2011), Neoscytalidium (Polizzi et al. 2009, Adesemoye et al. 2014, Mayorquin et al. 2016) and Sphaeropsis (Phillips et al. 2013).

China has a history of more than 4 000 years of citrus cultivation (Deng et al. 2008, Shen 2019) and is the world’s largest producer of citrus, with 37.92 M tons in 2018 (FAO 2018). Branch diseases including twig blight, branch dieback, bark rot, canker, crack and gummosis are commonly observed on citrus, especially in regions where stress factors such as frost and sunburn often occur. Resin (gummosis) caused by Diaporthe citri has been recorded as the most important fungal branch disease (Cai et al. 2011, Huang et al. 2013b), followed by Alternaria brown spot (dieback) caused by Alternaria alternata pathotype tangerine (Huang et al. 2012, Qin et al. 2012), anthracnose (twig blight and branch dieback) caused by Colletotrichum gloeosporioides (Cai et al. 2011, Huang et al. 2013a), and foot root caused by Phytophthora spp. (Cheng et al. 2004, Cai et al. 2011, Zhu et al. 2011). Species in other genera such as Cytospora, Diplodia, Dothidea, Macrophoma, Phoma, Phyllosticta and Sphaeropsis, have also been associated with citrus branch diseases (Chinese Academy of Agricultural Sciences 1960, Tai 1979). However, all fungal identifications were based on morphology or simply based on the symptoms before the 1990s and pathogenicity tests were lacking for most species (Tai 1979).

During 2017–2020, several surveys of citrus branch diseases were conducted in the major citrus production regions in China. The objectives of this study were to:

– identify the species of Botryosphaeriaceae associated with citrus branch diseases in China based on morphological traits and phylogenetic analysis;

– identify the dominant species associated with citrus branch diseases; and

– determine their pathogenicity.

MATERIALS AND METHODS

Disease symptoms, sample collection and fungal isolations

From 2017 to 2020, citrus branch disease samples with symptoms of canker, gummosis, twig blight and branch dieback (Fig. 1) were collected from the main citrus-producing regions in nine provinces of China, namely Chongqing, Fujian, Guangdong, Guangxi, Hunan, Jiangxi, Shaanxi, Shanghai and Zhejiang. The citrus species investigated and the number of samples collected would depend on the incidence of branch diseases in the orchard and region.

Fig. 1.

Fig. 1

Disease symptoms on citrus caused by Botryosphaeriaceae. a. Twig blight of Citrus reticulata; b. twig blight on Cocktail grapefruit; c. branch dieback of C. reticulata; d. death tree of C. reticulata; e. branch canker on C. reticulata; f. trunk canker of C. unshiu; g–h. gummosis on twig and trunk of Cocktail grapefruit; i. fungal fruitbody structures formed on dead branch of Cocktail grapefruit.

Fungal strains were isolated via two methods. Firstly, sporocarps visible on diseased tissue were transferred to a microtube containing sterile water to make a spore suspension. After dilution, 150 μL spore suspension was spread over the surface of water agar (WA) plates amended with 100 μg/mL ampicillin and 100 μg/mL streptomycin to suppress bacterial growth. After 24–36 h, germinating spores were retrieved and transferred onto potato dextrose agar plates (PDA, 200 g potatoes, 20 g glucose and 15 g agar/L water) with 100 μg/mL ampicillin and 100 μg/mL streptomycin (PDA-AS) and incubated at 25 °C. Axenic cultures were obtained by transferring a single colony onto PDA. Secondly, for samples lacking sporocarps, a tissue isolation method was used. A small section (about 3 × 3 mm) between the healthy and diseased tissue was aseptically cut and surface-sterilised in 70 % ethanol for 1 min, followed by 1 % NaClO solution for 1 min, and rinsed three times in sterile water. Tissue sections were dried on sterilised filter paper, placed on 1/2 PDA-AS plates and incubated at 25 °C. Axenic cultures were obtained by transferring single hyphal tips onto PDA. Specimens and isolates from this study were deposited in Zhejiang University, and ex-type cultures were deposited in the China General Microbiological Culture Collection Centre (CGMCC), Beijing, China.

DNA extraction, PCR amplification and sequencing

Isolates were grown on PDA plates and incubated at room temperature for 4–7 d. Surface mycelia were collected using a sterile scalpel blade and genomic DNA was extracted by the CTAB (Cetyl trimethylammonium bromide) method (Van Burik et al. 1998). Partial regions of four loci were amplified. The internal transcribed spacer region (ITS) was amplified with primers ITS1 and ITS4 (White et al. 1990). Part of the translation elongation factor 1-alpha gene (tef1) was amplified with primers EF1-688F (Alves et al. 2008) or EF1-728F and EF1-986R (Carbone & Kohn 1999). Part of the β-tubulin gene (tub2) was amplified with Bt2a and Bt2b (Glass & Donaldson 1995). Part of the DNA directed RNA polymerase II second largest subunit (rpb2) was amplified with RPB2-6F and fRPB2-7cR (Liu et al. 1999) or rpb2-lasF and rpb2-lasR (Cruywagen et al. 2017). All amplification reactions were performed in a total volume of 25 μL mixture consisted of 12.5 μL of 2 × Taq Master Mix (Dye Plus) (Vazyme), 9.5 μL ddH2O, 1 μL of each forward and reverse primer, and 1 μL DNA template. The amplification conditions consisted of an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. The PCR products were separated by agarose gel electrophoresis and sent to Qingke Biotechnology (Hangzhou, China) for Sanger DNA sequencing. The nucleotide sequences were assembled and edited with MEGA v. 7.0.26 (Kumar et al. 2016). Sequences obtained in this study were deposited in GenBank nucleotide database (http://www.ncbi.nlm.nih.gov; Table 1).

Table 1.

Details of Botryosphaeriaceae isolates studied.

Speciesa Isolate Location Collector Host Associated symptom GenBank Accession no.b
ITS tef1 tub2 rpb2
Botryosphaeria dothidea BE1 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MT772261 MT775839 MT775849 MW884107
(C. paradisi × C. reticulata)
BE2 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MT772262 MT775840 MT775850 MW884108
(C. paradisi × C. reticulata)
BE60 Chenggu, Shaanxi, China H.Y. Li & X.E. Xiao C. unshiu Trunk canker MW862113 MW884017 MW884086 MW884109
BE61 Changxing Island, Shanghai, China X.E. Xiao hybrid cv. Hongmeiren Twig dieback MW862116 MW884020 MW884087 MW884110
BE62 Chunan, Zhejiang, China H.Y. Li & X.E. Xiao C. unshiu Twig dieback MW862110 MW884014
BE63 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. reticulata Twig gummosis MW862111 MW884015
BE64 Quzhou, Zhejiang, China H.Y. Li & J.W. Lv hybrid cv. Cocktail grapefruit Twig gummosis MW862112 MW884016
(C. paradisi × C. reticulata)
BE65 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862114 MW884018
(C. paradisi × C. reticulata)
BE66 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862115 MW884019
(C. paradisi × C. reticulata)
BE67 Xiangshan, Zhejiang, China H.Y. Li C. unshiu Trunk canker MW862117 MW884021
BE68 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862118 MW884022
(C. paradisi × C. reticulata)
BE69 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. reticulata Twig gummosis MW862119 MW884023
BE72 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. reticulata Twig dieback MW881452 MW884024
BE73 Chenggu, Shaanxi, China H.Y. Li & X.E. Xiao C. unshiu Trunk gummosis MW862120 MW884025
BE75 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862121 MW884026
(C. paradisi × C. reticulata)
BE76 Hangzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. maxima Branch dieback MW862122 MW884027
BE77 Linhai, Zhejiang, China H.Y. Li C. unshiu Branch canker MW862123 MW884028
BE79 Hangzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. maxima Twig dieback MW862124 MW884029
BE81 Hangzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. maxima Branch dieback MW862125 MW884030
BE90 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW862126 MW884031
BE91 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. reticulata Twig gummosis MW862127 MW884032
BE92 Shaoyang, Hunan, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW862128 MW884033
BE93 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862129 MW884034
(C. paradisi × C. reticulata)
BE94 Linhai, Zhejiang, China H.Y. Li C. unshiu Branch gummosis MW862130 MW884035
BE95 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. reticulata Twig gummosis MW862131 MW884036
BE96 Chun’an, Zhejiang, China H.Y. Li & X.E. Xiao C. unshiu Branch dieback MW862132 MW884037
BE97 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862133 MW884038
(C. paradisi × C. reticulata)
BE98 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862134 MW884039
(C. paradisi × C. reticulata)
BE101 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW862135 MW884040
BE103 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW862136 MW884041
BE105 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW862137 MW884042
BE106 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW862138 MW884043
Botryosphaeria fabicerciana BE3 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MT772263 MT775841 MT775851 MW884111
(C. paradisi × C. reticulata)
BE78 Hangzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. maxima Twig dieback MW862139 MW884044 MW884088 MW884112
Botryosphaeria fabicerciana BE85 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862140 MW884045 MW884089 MW884113
(cont.) (C. paradisi × C. reticulata)
BE86 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit Twig gummosis MW862141 MW884046 MW884090 MW884114
(C. paradisi × C. reticulata)
Diplodia seriata BE4 Chenggu, Shaanxi, China H.Y. Li & X.E. Xiao C. unshiu Branch canker MW862142 MW884047 MW884091 MW884115
Dothiorella alpina BE17 Shimen, Hunan, China H.Y. Li & Y.T. Zeng C. unshiu Twig dieback MW862143 MW884048 MW884092 MW884116
Do. citrimurcotticola BE5 = CGMCC3.20392 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW880663 MW884166 MW884195 MW884140
BE6 Huangyan, Zhejiang, China H.K. Wang & X.E. Xiao C. maxima Twig dieback MW880664 MW884167 MW884196 MW884141
BE7 = CGMCC3.20393 Quzhou, Zhejiang, China H.Y. Li C. maxima Twig dieback MW880665 MW884168 MW884197 MW884142
BE8 * = CGMCC3.20394 Wanzhou, Chongqing, China H.Y. Li & X.E. Xiao hybrid cv. Murcott (C. reticulata × C. sinensis) Twig dieback MW880661 MW884164 MW884193 MW884138
BE9 = CGMCC3.20395 Wanzhou, Chongqing, China H.Y. Li & X.E. Xiao hybrid cv. Murcott Twig dieback MW880662 MW884165 MW884194 MW884139
(C. reticulata × C. sinensis)
BE71 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW880666 MW884169 MW884198 MW884143
Do. plurivora BE16 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao C. reticulata cv. Ponkan Twig dieback MT772270 MT775848 MT775858 MW884117
BE74 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW862144 MW884049 MW884093 MW884118
Lasiodiplodia citricola BE13 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao hybrid cv. Cocktail grapefruit (C. paradisi × C. reticulata) Branch gummosis MT772267 MT775845 MT775855 MW884119
BE38 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit (C. paradisi × C. reticulata) Twig gummosis MW862145 MW884050 MW884094 MW884120
BE45 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao C. unshiu Twig dieback MW862146 MW884051 MW884095 MW884121
BE83 Wanzhou, Chongqing, China H.Y. Li & X.E. Xiao hybrid cv. Hongmeiren Twig dieback MW862148 MW884053 MW884096 MW884122
BE89 Chenggu, Shaanxi, China H.Y. Li & X.E. Xiao C. unshiu Trunk canker MW862147 MW884052
BE99 Lishui, Zhejiang, China X.E. Xiao C. sinensis Twig dieback MW862149 MW884054
BE102 Xiangshan, Zhejiang, China H.Y. Li & X.E. Xiao C. unshiu Branch canker MW862150 MW884055 MW884097 MW884123
BE104 Lishui, Zhejiang, China X.E. Xiao hybrid Branch canker MW862151 MW884056 MW884098 MW884124
L. guilinensis BE31 * = CGMCC3.20378 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW880672 MW884175 MW884204 MW884149
BE59 = CGMCC3.20379 Linhai, Zhejiang, China H.Y. Li C. unshiu Branch gummosis MW880673 MW884176 MW884205 MW884150
L. huangyanensis BE33 * = CGMCC3.20380 Huangyan, Zhejiang, China X.E. Xiao & Q.B. Huang C. reticulata Twig dieback MW880674 MW884177 MW884206 MW884151
BE50 = CGMCC3.20381 Linhai, Zhejiang, China W.L. Li C. unshiu Branch canker MW880675 MW884178 MW884207 MW884152
BE111 Huangyan, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW880676 MW884179 MW884208 MW884153
L. iranensis BE27 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW880686 MW884189 MW884215 MW884160
BE30 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW880687 MW884190 MW884216 MW884161
BE36 Taizhou, Zhejiang, China X.E. Xiao & Q.B. Huang C. reticulata Trunk canker MW880688 MW884191 MW884217 MW884162
BE41 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao C. maxima Trunk canker MW862152 MW884057 MW884099 MW884125
BE42 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao C. maxima Trunk canker MW862153 MW884058 MW884100 MW884126
BE100 Lishui, Zhejiang, China X.E. Xiao C. maxima Trunk gummosis MW880684 MW884187 MW884213 MW884158
L. linhaiensis BE51 * = CGMCC3.20386 Linhai, Zhejiang, China W.L. Li C. unshiu Branch canker MW880677 MW884180 MW884209 MW884154
BE28 = CGMCC3.20383 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW880678 MW884181 MW884210 MW884155
BE34 = CGMCC3.20384 Huangyan, Zhejiang, China X.E. Xiao & Q.B. Huang C. reticulata Branch canker MW880679 MW884182 MW884211 MW884156
BE40 = CGMCC3.20385 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao hybrid cv. Cocktail grapefruit (C. reticulata × C. sinensis) Twig dieback MW880680 MW884183 MW884212 MW884157
BE43 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao C. reticulata Branch canker MW880681 MW884184
BE49 Linhai, Zhejiang, China W.L. Li C. unshiu Branch canker MW880682 MW884185
BE52 Linhai, Zhejiang, China W.L. Li C. unshiu Branch canker MW880683 MW884186
L. microconidia BE32 Huangyan, Zhejiang, China H.K. Wang & X.E. Xiao C. reticulata Branch canker MW880668 MW884171 MW884200 MW884145
BE35 Huangyan, Zhejiang, China X.E. Xiao & Q.B. Huang C. reticulata Branch canker MW880669 MW884172 MW884201 MW884146
BE80 Chun’an, Zhejiang, China H.Y. Li & X.E. Xiao C. grandis Trunk canker MW880670 MW884173 MW884202 MW884147
BE87 Chun’an, Zhejiang, China H.Y. Li & X.E. Xiao C. unshiu Trunk canker MW880671 MW884174 MW884203 MW884148
BE88 Chun’an, Zhejiang, China H.Y. Li & X.E. Xiao C. grandis Twig dieback MW880667 MW884170 MW884199 MW884144
L. ponkanicola BE44 * = CGMCC3.20388 Quzhou, Zhejiang, China H.K. Wang & X.E. Xiao C. reticulata Trunk canker MW880685 MW884188 MW884214 MW884159
L. pseudotheobromae BE10 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao C. reticulata Twig gummosis MT772264 MT775842 MT775852 MW884127
BE11 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MT772265 MT775843 MT775853 MW884128
BE12 Fuzhou, Jiangxi, China H.Y. Li & X.E. Xiao C. reticulata Twig dieback MT772266 MT775844 MT775854 MW884129
BE19 Guangzhou, Guangdong, China H.Y. Li C. reticulata Twig dieback MW862154 MW884059 MW884101 MW884130
BE22 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW862158 MW884063 MW884102 MW884131
BE23 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit (C. paradisi × C. reticulata) Twig gummosis MW862160 MW884065 MW884103 MW884132
BE24 Sihui, Guangdong, China H.Y. Li C. reticulata Twig dieback MW862155 MW884060
BE26 Sihui, Guangdong, China H.Y. Li C. reticulata Twig dieback MW862156 MW884061
BE29 Sihui, Guangdong, China H.Y. Li C. reticulata Twig dieback MW862157 MW884062
BE37 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. sinensis Twig dieback MW862159 MW884064
BE39 Quzhou, Zhejiang, China H.Y. Li & X.E. Xiao hybrid cv. Cocktail grapefruit (C. paradisi × C. reticulata) Twig gummosis MW862161 MW884066
BE46 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862162 MW884067
BE47 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862163 MW884068
BE48 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862164 MW884069
BE53 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862165 MW884070
BE54 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862166 MW884071
BE55 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862167 MW884072
BE56 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862168 MW884073
BE57 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862169 MW884074
BE58 Linhai, Zhejiang, China W.L. Li C. unshiu Trunk canker MW862170 MW884075
BE70 Wanzhou, Chongqing, China H.Y. Li & X.E. Xiao C. limon Twig dieback MW862171 MW884076
BE82 Fuzhou, Jiangxi, China H.Y. Li & X.E. Xiao C. reticulata Twig dieback MW862172 MW884077
BE84 Chun’an, Zhejiang, China H.Y. Li & X.E. Xiao C. unshiu Trunk canker MW862173 MW884078
BE107 Yongchun, Fujian, China X.E. Xiao C. reticulata Twig dieback MW862174 MW884079
BE109 Yongchun, Fujian, China X.E. Xiao C. sinensis Twig dieback MW862175 MW884080
BE110 Yongchun, Fujian, China X.E. Xiao C. reticulata Twig dieback MW862176 MW884081
L. theobromae BE20 Sihui, Guangdong, China H.Y. Li C. reticulata Twig dieback MW862177 MW884082 MW884104 MW884133
BE21 Sihui, Guangdong, China H.Y. Li C. reticulata Twig dieback MW862178 MW884083 MW884105 MW884134
BE25 Guilin, Guangxi, China H.Y. Li & X.E. Xiao C. reticulata Twig dieback MW862179 MW884084 MW884106 MW884135
BE108 Yongchun, Fujian, China X.E. Xiao C. sinensis Twig dieback MW862180 MW884085
Neodeightonia subglobosa BE14 Xiangshan, Zhejiang, China H.Y. Li & B. Liu C. unshiu Trunk gummosis MT772268 MT775846 MT775856 MW884136
Neofusicoccum parvum BE15 Xiangshan, Zhejiang, China X.E. Xiao C. unshiu Branch gummosis MT772269 MT775847 MT775857 MW884137
Sphaeropsis linhaiensis BE18 * = CGMCC3.20382 Linhai, Zhejiang, China H.Y. Li C. unshiu Twig dieback MW880689 MW884192 MW884218 MW884163

a Species names in bold represent new species described in this study.

b ITS, internal transcribed spacer region and intervening 5.8S nrRNA gene; tef1, translation elongation factor 1-alpha; tub2, β-tubulin; rpb2, DNA-directed RNA polymerase II second largest subunit.

* Isolates represent ex-type.

Phylogenetic analyses

Sequences of the ITS and tef1 locus for all the isolates obtained in this study were generated and blasted against the NCBIs GenBank nucleotide datasets (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to obtain an initial identification. Representative isolates were selected for sequencing of tub2 and rpb2 loci and further phylogenetic analyses. Sequences of ex-type strains closely related to the Botryosphaeriaceae isolates studied here were downloaded from NCBI and used for phylogenetic analyses (Table 2). Sequence alignments of each of the ITS, tef1, tub2 and rpb2 loci were initially aligned by using MAFFT v. 7 online service (https://mafft.cbrc.jp/alignment/server/index.html) (Katoh et al. 2019), with iterative refinement methods (FFT-NS-i), and then edited manually with MEGA v. 7.0.26 software. Aligned datasets and phylogenetic trees for the individual genes and combined alignments were deposited in TreeBASE (http://treebase.org; study number S28083).

Table 2.

Isolates from other studies used in the phylogenetic analyses.

Species Isolate numbersa Host Location Collector GenBank accession numbersb
ITS tef1 tub2 rpb2
Botryosphaeria agaves CBS 133992 = MFLUCC11-0125 * Agave sp. Thailand R. Phookamsak JX646791 JX646856 JX646841
MFLUCC 10-0051 Agave sp. Thailand P. Chomnunti JX646790 JX646855 JX646840
Botryosphaeria corticis CBS 119047 * Vaccinium corymbosum USA P.V. Oudemans DQ299245 EU017539
ATCC 22927 Vaccinium sp. USA R.D. Millholland DQ299247 EU673291 EU673108
Botryosphaeria dothidea CBS 115476 = CMW 8000 * Prunus sp. Switzerland B. Slippers AY236949 AY236898 AY236927 EU339577
CBS 110302 Vitis vineifera Portugal A.J.L. Phillips AY259092 AY573218 EU673106
CBS 145971 = CPC 29048 Grevillea sp. Australia P.W. Crous MT587332 MT592034 MT592470
Botryosphaeria fabicerciana CBS 127194 = CMW 27094 * Eucalyptus sp. China M.J. Wingfield HQ332197 HQ332213 KF779068 MF410137
CERC 2948 Eucalyptus sp. China M.J. Wingfield KX277983 KX278088 KX278193 MF410132
Botryosphaeria kuwatsukai CBS 135219 = PG2 * Malus domestica China C.S. Wang KJ433388 KJ433410
LSP5 Pyrus sp. China C.S. Wang KJ433395 KJ433417
Botryosphaeria qingyuanensis CERC 2946 = CGMCC 3.18742 * Eucalyptus hybrid China S.F. Chen & G.Q. Li KX278000 KX278105 KX278209 MF410151
CERC 2947 = CGMCC 3.18744 Eucalyptus hybrid China S.F. Chen & G.Q. Li KX278001 KX278106 KX278210 MF410152
Botryosphaeria ramosa CBS 122069 = CMW 26167 * Eucalyptus camaldulensis Australia T.I. Burgess EU144055 EU144070 KF766132
CGMCC 3.18006 Myrtaceae China KX197072 KX197092 KX197099
Botryosphaeria scharifii CBS 124703 = IRAN 1529C * Mangifera indica Iran J. Abdollahzadeh JQ772020 JQ772057
CBS 124702 = IRAN 1543C Mangifera indica Iran J. Abdollahzadeh & A. Javadi JQ772019 JQ772056
Diplodia africana CBS 120835 = CPC 5908 * Prunus persica South Africa U. Damm KF766155 KF766397 KF766129
STE-U 5946 Prunus persica South Africa U. Damm EF445344 EF445383
Diplodia afrocarpi CBS 131681 = CMW 35506 Afrocarpus falcatus, healthy twigs South Africa E.M. Cruywagen MT587333 MT592035 MT592471
Diplodia agrifolia CBS 124.30 Ulmus sp. USA KX464087 KX464557 KX464783 KX463953
Diplodia allocellula CBS 130408 = CMW 36468 * Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239397 JQ239384 JQ239378
CMW 36470 Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239399 JQ239386 JQ239380
Diplodia arengae MFLU 17-2769 = XTBG28 * Arenga hookeriana China D.N. Wanasinghe MG762771 MG762774 MG783039
Diplodia bulgarica CBS 124254 * Malus sylvestris Bulgaria S.G. Bobev GQ923853 GQ923821
CBS 124135 Malus sylvestris Bulgaria S.G. Bobev GQ923852 GQ923820
Diplodia citricarpa CBS 124715 = CJA 131 = IRAN 1578C * Citrus sp., twigs Iran J. Abdollahzadeh & A. Javadi KF890207 KF890189 KX464784
Diplodia corticola CBS 112549 = CAP 134 * Quercus suber Portugal A. Alves AY259100 AY573227 DQ458853
CBS 112546 Quercus ilex Spain AY259090 EU673310 EU673117 KX463954
Diplodia crataegicola MFLU 15-1311 * Crataegus sp. Italy KT290244 KT290248 KT290246
Diplodia cupressi CBS 168.87 * Cupressus sempervirens Israel Z. Solel DQ458893 DQ458878 DQ458861
CBS 261.85 Cupressus sempervirens Israel Z. Solel DQ458894 DQ458879 DQ458862
Diplodia eriobotryicola CBS 140851 = BN-21 * Eriobotrya japonica Spain E. Gonzalez-Domýnguez KT240355 KT240193 MG015806
Diplodia estuarina CMW 41363 Rhizophora mucronata South Afric J.A. Osorio & Jol. Roux. KP860829 KP860674 KP860752
CMW 41230 Rhizophora mucronata South Afric J.A. Osorio & Jol. Roux. KP860830 KP860675 KP860753
Diplodia fraxini CBS 136010 * Fraxinus angustifolia Portugal A. Deidda KF307700 KF318747 MG015807
CBS 136011 Fraxinus angustifolia Italy B.T. Linaldeddu KF307711 KF318748 MG015808
Diplodia galiicola MFLU15-1310 * Galium sp. Italy E. Camporesi KT290245 KT290249 KT290247
Diplodia gallae CBS 211.25 Quercus sp., fruit KX464090 KX464564 KX464795
CBS 212.25 Quercus sp., gall KX464091 KX464565 KX464796
Diplodia malorum CBS 124130 * Malus sylvestris Portugal A.J.L. Phillips GQ923865 GQ923833
BN-37 Eriobotrya japonica Spain KT240360 KT240198
Diplodia mutila CBS 112553 = CAP 062 * Vitis vinifera Portugal A.J.L. Phillips AY259093 AY573219 KY554743
Diplodia neojuniperi CPC 22753 = B0031 * Juniperus chinensis Thailand T. Trakunyingcharoen KM006431 KM006462
CPC 22754 = B0032 Juniperus chinensis Thailand T. Trakunyingcharoen KM006432 KM006463
Diplodia olivarum CBS 121887 = CAP 254 * Olea europaea Italy C. Lazzizera EU392302 EU392279 HQ660079
IMI 390972 Carob tree Italy HM028640 HQ660078 HQ660080
Diplodia pseudoseriata CBS 124906 * Blepharocalyx salicifolius Uruguay C. Pérez EU080927 EU863181 MG015820
Diplodia quercivora CBS 133852 * Quercus canariensis Tunisia B. T. Linaldeddu JX894205 JX894229 MG015821
MEAN 1017 Quercus suber Portugal H. Braganca KU311198 KU311201
Diplodia rosulata CBS 116470 * Prunus africana Ethiopia A. Gure EU430265 EU430267 EU673132
CBS 116472 Prunus africana Ethiopia A. Gure EU430266 EU430268 EU673131
Diplodia sapinea CBS 393.84 * Pinus nigra Netherlands H.A. van der Aa DQ458895 DQ458880 DQ458863
CBS109726 = CMW 04880 Pinus patula South Africa M.J. Wingfield KX464094 KX464568 KX464800 KX463956
Diplodia scrobiculata CBS 118110 * Pinus resinosa USA M.A. Palmer AY253292 AY624253 AY624258 KX463959
Diplodia seriata CBS 112555 = CAP 063 * Vitis vinifera Italy A.J.L. Phillips AY259094 AY573220 DQ458856
CBS 117.82 Rubus sp., dead stem Italy H.A. van der Aa KX464108 KX464598 KX464834 KX463964
Diplodiasp. 1 CBS 678.88 Quercus suber Spain J. Luque AY259104 GU799459 GU799458
UCD1275So Grape vine USA GU799471 GU799468 GU799465
Diplodia subglobosa CBS 124133 = JL 453 * Lonicera nigra Spain J. Luque GQ923856 GQ923824 MT592576
CBS 124132 = JL 375 Fraxinus excelsior Spain J. Luque DQ458887 DQ458871 DQ458852
Diplodia tsugae CBS 418.64 = IMI 197143 * Tsuga heterophylla Canada A. Funk DQ458888 DQ458873 DQ458855
Dothiorella acacicola CBS 141295 = CPC 26349 * Acacia mearnsii France P.W. Crous & M.J. Wingfield KX228269 KX228376
Dothiorella acericola KUMCC 18-0137 * Acer palmatum, dead hanging twigs China R. Phookamsak MK359449 MK361182
HNXX032 Ziziphus jujuba, branch China R. Zang KY385661 KY393212 KY393178
Dothiorella alpina CGMCC 3.18001 * Platycladus orientalis China W. He & J.R. Wu; det. Y. Zhang KX499645 KX499651
Dothiorella brevicollis CBS 130411 = CMW 36463 * Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239403 JQ239390 JQ239371
CMW 36464 Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239404 JQ239391 JQ239372
Dothiorella capri-amissi CBS 121763 = CMW 25403 * Acacia erioloba South Africa F.J.J. van der Walt & G.J. Marais EU101323 EU101368 KX464850
CMW 25404 Acacia erioloba South Africa F.J.J. van der Walt & G.J. Marais EU101324 EU101369
Dothiorella casuarini CBS 120688 = CMW 4855 * Casuarina sp. Australia M.J. Wingfield DQ846773 DQ875331 KX463970
CBS 120690 = CMW 4857 Casuarinasp. Australia M.J. Wingfield DQ846774 DQ875333
Dothiorella citricola CBS 124729 = ICMP 16828 * Citrus sinensis New Zealand S.R. Pennycook, P.R. Johnston & B.C. Paulus EU673323 EU673290 KX464853 KX463971
CBS 124728 = ICMP 16827 Citrus sinensis New Zealand S.R. Pennycook, P.R. Johnston & B.C. Paulus EU673322 EU673289 KX464852
Dothiorella diospyricola CBS 145972 = CPC 34653 * Diospyros mespiliformis South Africa P.W. Crous MT587398 MT592110 MT592581
Dothiorella dulcispinae CBS 130413 = CMW 36460 * Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239400 JQ239387 JQ239373
CMW 36462 Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239402 JQ239389 JQ239375
Dothiorella eriobotryae CBS 140852 = CPC 29679 = BN 81 * Eriobotrya japonica, branch canker Spain E. Gonzalez-Domýnguez KT240287 KT240262 MT592582
Dothiorella heterophyllae CMW46458 Acacia heterophylla La Réunion M.J. Wingfield MN103794 MH548348 MH548324
Dothiorella iranica CBS 124722 = IRAN 1587C * Olea europea Iran A. Javadi KC898231 KC898214 KX464856
MFLUCC 15-0656 Paliurus Italy E. Camporesi KX765302 KX765303
Dothiorella koae CMW48017 Acacia heterophylla La Réunion M.J. Wingfield MH447652 MH548338 MH548327
Dothiorella lampangensis MFLUCC 18-0232 * Rutaceae, fallen fruit pericarp Thailand S.C. Jayasiri MK347758 MK340869 MK412874
Dothiorella longicollis CBS 122068 = CMW 26166 * Lysiphyllum cunninghamii Australia T.I. Burgess & M.J. Wingfield EU144054 EU144069 KF766130 KX463972
CBS 122066 = CMW 26166 Terminalia sp. Australia T.I. Burgess & M.J. Wingfield EU144052 EU144067 KX464857
Dothiorella magnoliae CFCC 51563 * Magnolia grandiflora China C.J. You KY111247 KY213686
CFCC 51564 Magnolia grandiflora China C.J. You KY111248 KY213687
Dothiorella mangifericola CBS 124727 = IRAN 1584C * Mangifera indica Iran J. Abdollahzadeh & A. Javadi KC898221 KC898204 KX463973
IRAN 1545C Mangifera indica Iran J. Abdollahzadeh & A. Javadi KC898223 KC898206
Dothiorella moneti MUCC 505 = WAC 13154 * Acacia rostellifera Australia K.M. Taylor EF591920 EF591971 EF591954
MUCC 507 Acacia rostellifera Australia K.M. Taylor EF591922 EF591973 EF591956
Dothiorella plurivora CBS 124724 = IRAN 1557C * Citrus sp. Iran J. Abdollahzadeh & A. Javadi KC898225 KC898208 KX464874
CBS 124725 Prunus armeniaca Iran J. Abdollahzadeh & A. Javadi KC898225 KC898213 KX464875
Dothiorella pretoriensis CBS 130404 = CMW 36480 * Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239405 JQ239392 JQ239376
CMW 36481 Acacia karroo South Africa F. Jami & M. Gryzenhout JQ239406 JQ239393 JQ239377
Dothiorella prunicola CBS 124723 = CAP 187 * Prunus dulcis Portugal E. Diogo EU673313 EU673280
Dothiorella reunionis CMW46457 * Acacia heterophylla La Réunion M.J. Wingfield MH447649 MH548347
Dothiorella santali MUCC 509 = WAC 13155 * Santalum acuminatum Australia K.M. Taylor EF591924 EF591975 EF591958
MUCC 508 Santalum acuminatum Australia K.M. Taylor EF591923 EF591974 EF591957
Dothiorella sarmentorum IMI 63581b * Ulmus sp. England E.A. Ellis AY573212 AY573235
CBS 115038 Malus pumila Netherlands A.J.L. Phillips AY573206 AY573223 EU673101
Dothiorella sp. 1 CBS 121783 = CMW 25432 = CAMS 1187 Acacia mearnsii South Africa F.J.J. van der Walt & R.N. Heath EU101333 EU101378 KX464859
CBS 121784 = CMW 25430 = CAMS 1185 Acacia mearnsii South Africa F.J.J. van der Walt & R.N. Heath EU101331 EU101376 KX464860
Dothiorella striata CBS 124731= ICMP 16824 * Citrus sinensis New Zealand S.R. Pennycook, P.R. Johnston & B.C. Paulus EU673321 EU673288 EU673143 KX463976
CBS 124730 = ICMP 16819 Citrus sinensis New Zealand S.R. Pennycook, P.R. Johnston & B.C. Paulus EU673320 EU673287 EU673142
Dothiorella tectonae MFLUCC 12-0382 = MD-2014 * Tectona grandis Thailand M. Doilom KM396899 KM409637 KM510357
Dothiorella thailandica MFLUCC 11-0438 * Bamboo culm Thailand D.Q. Dai JX646796 JX646861 JX646844
Dothiorella thripsita CBS 125445 = BRIP 51876 * Acacia harpophylla Australia D.J. Tree & C.E.C. Tree FJ824738 KJ573639 KJ577550 KX463977
Dothiorella ulmacea CBS 138855 = CPC 24416 * Ulmus laevis Germany R.K. Schumacher KR611881 KR611910 KR611909
CPC 24945 Ulmus laevis Germany R.K. Schumacher KR611882 KR857697
Dothiorella uruguayensis CBS 124908 = CMW 26763 =UY672 * Hexachlamis edulis Uruguay C.A. Pérez EU080923 EU863180 KX464886
Dothiorella vinea-gemmae DAR 81012 = B116-3 * Vitis vinifera Australia N. Wunderlich KJ573644 KJ573641
Dothiorella viticola CBS 117009 * Vitis vinifera cv. Garnatxa Negra Spain J. Luque & S. Martos AY905554 AY905559 EU673104 DQ677985
GAR09 Vitis sp. French KT595694 KX098285 KT595695
Dothiorella yunnana CGMCC 3.17999 * Camellia sp. China W. He & J.R. Wu; det. Y. Zhang KX499643 KX499649
CGMCC 3.18000 Camellia sp. China W. He & J.R. Wu; det. Y. Zhang KX499644 KX499650
Lasiodiplodia acaciae CBS 136434 = CPC 20820 * Acacia sp., leaf spot Indonesia M.J. Wingfield MT587421 MT592133 MT592613 MT592307
Lasiodiplodia americana CERC 1961 = CFCC 50065 * Pistachia vera USA T.J. Michailides KP217059 KP217067 KP217075 MF410161
CERC 1960 = CFCC 50064 Pistachia vera USA T.J. Michailides KP217058 KP217066 KP217074 MF410162
Lasiodiplodia aquilariae CGMCC 3.18471 * Aquilaria crassna Laos X. Sun KY783442 KY848600 KY848562
Lasiodiplodia avicenniae CMW 41467 * Avocennia marina South Africa J.A. Osorio & J. Roux KP860835 KP860680 KP860758 KU587878
LAS 199 Avocennia marina South Africa J.A. Osorio & J. Roux KU587957 KU587947 KU587868 KU587880
Lasiodiplodia brasiliense CMM 4015 * Mangifera indica Brazil M.W. Marques JX464063 JX464049
IBL 344 Adansonia madagascariensis Madagascar KT151808 KT151802 KT151805
Lasiodiplodia bruguierae CMW 41470* Bruguiera gymnorrhiza South Africa J.A. Osorio & J. Roux KP860833 KP860678 KP860756 KU587875
CMW 41614 Bruguiera gymnorrhiza South Africa J.A. Osorio & J. Roux KP860834 KP860679 KP860757 KU587877
Lasiodiplodia cinnamomi CFCC 51997 * Cinnamomum camphora China N. Jiang MG866028 MH236799 MH236797 MH236801
CFCC 51998 Cinnamomum camphora China N. Jiang MG866029 MH236800 MH236798 MH236802
Lasiodiplodia citricola CBS 124707 = IRAN 1522C * Citrus sp. Iran J. Abdollahzadeh & A. Javadi GU945354 GU945340 KP872405 KU696351
CBS124706 = IRAN 1521C Citrus sp. Iran A. Shekari GU945353 GU945339 KP872406 KU696350
Lasiodiplodia crassispora CBS 118741 = WAC12533 * Santalum album Australia T.I. Burgess & B. Dell DQ103550 EU673303 KU887506 KU696353
CMM 4585 MG954354 MG979520 MG979552 MG979561
Lasiodiplodia euphorbicola CMM 3609 * Jatropha curcas Brazil A.R. Machado & O.L. Pereira KF234543 KF226689 KF254926
CMW 33350 Adansonia digitata Botswana KU887149 KU887026 KU887455 KU696346
Lasiodiplodia gilanensis CBS 124704 = IRAN1523C= UCCE 940B * Citrus sp., fallen twigs Iran J. Abdollahzadeh & A. Javadi KX906851 KX906853 KX906849 KU696357
CBS 124705 = IRAN 1501C Citrus sp., fallen twigs Iran J. Abdollahzadeh & A. Javadi GU945352 GU945341 KP872412 KU696356
Lasiodiplodia gonubiensis CBS 115812 = CMW 14077 * Syzygium cordatum South Africa D. Pavlic AY639595 DQ103566 DQ458860 KU696359
CMW 46621 = MTU 56 Syzygium cordatum South Africa D. Pavlic KY052944 KY024623 KY000126
Lasiodiplodia gravistriata CMM 4564 * Anacardium humile Brazil M.S.B. Netto KT250949 KT250950
CMM 4565 Anacardium humile Brazil M.S.B. Netto KT250947 KT266812
Lasiodiplodia hormozganensis CBS 124709 = IRAN 1500C * Olea sp. Iran J. Abdollahzadeh & A. Javadi GU945355 GU945343 KP872413 KU696361
CBS 124708 = IRAN 1498C Mangifera indica Iran J. Abdollahzadeh & A. Javadi GU945356 GU945344 KP872414 KU696360
Lasiodiplodia indica IBP 1 * Angiospermous tree India I.B. Prasher & G. Singh KM376151
Lasiodiplodia iranensis CBS 124710 = IRAN 1520C * Salvadora persica Iran J. Abdollahzadeh & A. Javadi GU945348 GU945336 KU887516 KU696363
CBS 124711 = IRAN 1502C = CMM 4603 Juglans sp. Iran A. Javadi GU945347 GU945335 MG979537
Lasiodiplodia laeliocattleyae CBS 167.28 * Laeliocattleya Italy C. Sibilia KU507487 KU507454
CMM 4724 Vitis vinifera Brazil MG954343 MG979508 MG979541
Lasiodiplodia lignicola CBS 134112= MFLUCC 11-0435 * Dead wood Thailand A.D. Ariyawansa JX646797 KU887003 KT852958 KU696364
Lasiodiplodia macrospora CMM 3833 * Jatropha curcas Brazil A.R. Machado & O.L. Pereira KF234557 KF226718 KF254941
Lasiodiplodia mahajangana CBS 124925 = CMW 27801 * Terminalia catappa Madagascar J. Roux FJ900595 FJ900641 FJ900630 KU696365
CMW 27818 Terminalia catappa Madagascar J. Roux FJ900596 FJ900642 FJ900631
Lasiodiplodia margaritacea CBS 122519 = CMW 26162 * Adansonia gibbosa Australia T.I. Burgess & M.J. Wingfield EU144050 EU144065 KX464903 KU696367
CBS 122065 Adansonia gibbosa Australia T.I. Burgess & M.J. Wingfield EU144051 EU144066
Lasiodiplodia mediterranea CBS 137783 * Holm oak Italy B.T. Linaldeddu KJ638312 KJ638331 KU887521 KU696368
CBS137784 Grapevine Italy S. Serra KJ638311 KJ638330 KU887522 KU696369
Lasiodiplodia microconidia CGMCC 3.18485 * Aquilaria crassna Laos X. Sun KY783441 KY848614 KY848561
Lasiodiplodia parva CBS 456.78 * Cassava-field soil Colombia O. Rangel EF622083 EF622063 KU887523 KU696372
CBS 494.78 Cassava-field soil Colombia O. Rangel EF622084 EF622064 EU673114 KU696373
Lasiodiplodia plurivora CBS 120832 = STE-U5803 * Prunus salicina South Africa F. Halleen EF445362 EF445395 KP872421 KU696374
CBS 121103 = STE-U4583 Vitis vinifera South Africa F. Halleen AY343482 EF445396 KP872422 KU696375
Lasiodiplodia pontae CMW 1277 = IBL12 * Spondias purpurea Brazil J.S. Lima & F.C.O. Freire KT151794 KT151791 KT151797
Lasiodiplodia pseudotheobromae CBS 116459 * Gmelina arborea Costa Rica J. Carranza & Velásquez EF622077 EF622057 EU673111 KU696376
CMM 3887 Jatropha curcas Brazil A.R. Machado KF234559 KF226722 KF254943
Lasiodiplodia rubropurpurea CBS 118740 = CMW 14700 = WAC 12535 * Eucalyptus grandis Australia T.I. Burgess & G. Pegg DQ103553 DQ103571 KU887529 KU696380
WAC 12536 = CMW 15207 Eucalyptus grandis Australia T.I. Burgess & G. Pegg DQ103554 DQ103572 KP872425 KU696381
Lasiodiplodia subglobosa CMM 3872 * Jatropha curcas Brazil A.R. Machado & O.L. Pereira KF234558 KF226721 KF254942
CMM 4046 Jatropha curcas Brazil A.R. Machado & O.L. Pereira KF234560 KF226723 KF254944
Lasiodiplodia syzygii MFLUCC 19-0219.1 = GUCC 9719.1 * Syzygium samarangense Thailand Q. Zhang MT990531 MW016943 MW014331
GUCC 9719.3 Syzygium samarangense Thailand Q. Zhang MW081992 MW087102 MW087105
Lasiodiplodia thailandica CPC 22795 * Mangifera indica Thailand T. Trakunyingcharoen KJ193637 KJ193681
BJFU DZP160123-13 Albizia chinensis China Z.P. Dou & Z.C. Liu KY676789 KY676798 KY751301 KY751298
Lasiodiplodia theobromae CBS 164.96 * fruit along coral reef coast Papua New Guinea A. Aptroot AY640255 AY640258 KU887532 KU696383
CBS 111530 Leucospermum sp. USA J.E. Taylor EF622074 EF622054 KU887531 KU69638
CBS 124.13 USA J.J. Taubenhaus DQ458890 DQ458875 DQ458858 KY472887
Lasiodiplodia tropica CGMCC 3.18477 * Aquilaria crassna Laos X. Sun KY783454 KY848616 KY848540 KY848574
Lasiodiplodia venezuelensis CBS 118739 = CMW 13511 = WAC 12539 * Acacia mangium Venezuela S. Mohali DQ103547 EU673305 KU887533 KU696384
CBS 129757 Acacia mangium Venezuela S. Mohali JX545102 JX545122 JX545142
Lasiodiplodia viticola CBS 128313 = UCD 2553AR * Vitis vinifera USA R.D. Cartwright & W.D. Gubler HQ288227 HQ288269 HQ288306 KU696385
CBS 128315 = UCD 2604MO Vitis vinifera USA K. Striegler & W.D. Gubler HQ288228 HQ288270 HQ288307 KU696386
Lasiodiplodia vitis CBS 124060 * Vitis vinifera Italy S. Burruano KX464148 KX464642 KX464917
Neodeightonia licuriensis COAD 1780 * Syagrus coronata Brazil O.L. Pereira KP165429 KP165430 KP165431
Neodeightonia microspora MFLUCC 11-0483 bamboo Thailand D.Q. Dai KU940110
MFLUCC 11-0504 bamboo Thailand D.Q. Dai KU940111
Neodeightonia palmicola MFLUCC10-0822 * Arenga westerhoutii Thailand J.K. Liu HQ199221
FAFU 002 Caryota mitis China MK203813 MK208460
Neodeightonia phoenicum CBS 122528 * Phoenix sp. Spain F. Garcia EU673340 EU673309 EU673116 KX463999
CBS 169.34 Phoenix dactylifera USA H.S. Fawcett EU673338 EU673307 EU673138
Neodeightonia planchoniae MFLUCC 17-2427 Planchonia sp. Thailand S.C. Jayasiri MK347755
Neodeightonia rattanica MFLUCC 15-0712 * Calamus sp. Thailand S. Konta KX646357 KX646360
MFLUCC 15-0313 Calamus sp. Thailand S. Konta KX646358 KX646361
Neodeightonia rattanicola MFLUCC 15-0319 * Calamus sp. Thailand S. Konta KX646359 KX646362
Neodeightonia subglobosa CBS 448.91 * Bambusa arundinacea Sierra Leone F.C. Deighton EU673337 EU673306 EU673137
MFLUCC 11-0607 bamboo Thailand D.Q. Dai KU940113
Neofusicoccum algeriense CBS 137504 = ALG1 * Vitis vinifera Algeria A. Berraf-Tebbal KJ657702 KJ657715
ALG9 Vitis vinifera Algeria A. Berraf-Tebbal KJ657704 KJ657721
Neofusicoccum andinum CBS 117453 = CMW 13455 * Eucalyptus sp. Venezuela S. Mohali AY693976 AY693977 KX464923 KX464002
CBS 117452 = CMW 13446 Eucalyptus sp. Venezuela S. Mohali DQ306263 DQ306264 KX464922 KX464001
Neofusicoccum arbuti CBS 116131 * Arbutus menziesii USA A. Rossman AY819720 KF531793 KX464003
CBS 116575 Arbutus menziesii USA M. Elliott KX464155 KX464650 KX464927
Neofusicoccum australe CMW 6837 * Acacia sp. Australia M.J. Wingfield AY339262 AY339270 AY339254 EU339573
C1.2 Arctostphylos glauca USA L. Drake-Schultheis MH777002 MH754929
Neofusicoccum batangarum CBS 124924 = CMW 28363 * Terminalia catappa Cameroon D. Begoude & J. Roux FJ900607 FJ900653 FJ900634 FJ900615
OB45 Opuntia ficus-indica Italy MG609042 MG609076 MG609059
Neofusicoccum brasiliense CMM 1338 * Mangifera indica Brazil M.W. Marques JX513630 JX513610 KC794031
CMM 1269 Mangifera indica Brazil M.W. Marques JX513629 JX513609 KC794032
Neofusicoccum cordaticola CBS 123634 = CMW 13992 * Syzygium cordatum South Africa D. Pavlic EU821898 EU821868 EU821838 EU821928
CBS123635 = CMW 14056 Syzygium cordatum South Africa D. Pavlic EU821903 EU821873 EU821843 EU821933
Neofusicoccum cryptoaustrale CBS 122813 = CMW 23785 * Eucalyptus tree South Africa H.M. Maleme FJ752742 FJ752713 FJ752756 KX464014
Neofusicoccum eucalypticola CBS 115679 = CMW 6539 * Eucalyptus sp. Australia M.J. Wingfield AY615141 AY615133 AY615127
CBS 6539 = CMW 6217 Eucalyptus sp. Australia M.J. Wingfield AY615143 AY615135 AY615125
Neofusicoccum eucalyptorum CBS 115791 = CMW 10125 * Eucalyptus grandis South Africa H. Smith AF283686 AY236891 AY236920
CAA 518 Eucalyptus globulus Portugal KX871883 KX871839 KX871776
Neofusicoccum grevilleae CBS 129518 = CPC 16999 * Grevillea aurea Australia P.W. Crous & R.G. Shivas JF951137
Neofusicoccum hellenicum CERC 1947 = CFCC 50067 * Pistacia vera Greece T.J. Michailides KP217053 KP217061 KP217069
CERC 1948 = CFCC 50068 Pistacia vera Greece T.J. Michailides KP217054 KP217062 KP217070
Neofusicoccum kwambonambiense CBS 123639 = CMW 14023 * Syzygium cordatum South Africa D. Pavlic EU821900 EU821870 EU821840 EU821930
CBS 123641 = CMW 14140 Syzygium cordatum South Africa D. Pavlic EU821919 EU821889 EU821859 EU821949
Neofusicoccum lumnitzerae CMW 41469 * Lumnitzera racemosa South Africa J.A Osorio & Jol. Roux KP860881 KP860724 KP860801 KU587925
CMW 41228 Lumnitzera racemosa South Africa J.A Osorio & Jol. Roux KP860882 KP860725 KP860803 KU587926
Neofusicoccum luteum CBS 562.92 = ATCC 58193* Actinidia deliciosa New Zealand S.R. Pennycook KX464170 KX464690 KX464968 KX464020
BRIP 5016 Persea americana USA MH057191 MH102254
Neofusicoccum macroclavatum CBS 118223 = WAC 12444 * Eucalyptus globulus Australia T.I. Burgess DQ093196 DQ093217 DQ093206 KX464022
WAC 12446 Eucalyptus globulus Australia T.I. Burgess DQ093197 DQ093218 DQ093207
Neofusicoccum mangiferae CBS 118531 = CMW 7024 * Mangifera indica Australia G.I. Johnson AY615185 DQ093221 AY615172
CBS 118532 = CMW 7797 Mangifera indica Australia G.I. Johnson AY615186 DQ093220 AY615173 KX464023
Neofusicoccum mangroviorum CMW 41365 * Avicennia marina South Africa J.A. Osorio KP860859 KP860702 KP860779 KU587905
CMW 42481 Bruguiera gymnorrhiza South Afric J.A. Osorio KP860848 KP860692 KP860770 KU587895
Neofusicoccum mediterraneum CBS 12718 = PD 312 * Eucalyptus sp. Greece P.W. Crous, M.J. Wingfield & A.J.L. Phillips GU251176 GU251308 GU251836 KX464024
Neofusicoccum microconidium CGMCC 3.18750 = CERC 3497 Eucalyptus urophylla × E. grandis China S.F. Chen & G.Q. Li KX278053 KX278158 KX278262 MF410203
Neofusicoccum nonquaesitum CBS 126655 = PD 484 * Umbellularia californica USA F.P. Trouillas GU251163 GU251295 GU251823 KX464025
PD301 Vaccinium corymbosum Chile E.X. Briceño, J.G. Espinoza & B. A.Latorre GU251164 GU251296 GU251824
Neofusicoccum occulatum CBS 128008 = MUCC 227 * Eucalyptus grandis Australia T.I. Burgess EU301030 EU339509 EU339472 EU339558
MUCC 286 = WAC 12395 Eucalyptus pellita Australia T.I. Burgess EU736947 EU339511 EU339474 EU339560
Neofusicoccum parvum CMW 9081 = ATCC 58191* Populus nigra New Zealand S.R. Pennycook AY236943 AY236888 AY236917 EU821963
CBS 110301 Vitis vinifera Portugal A.J.L. Phillips AY259098 AY573221 EU673095
Neofusicoccum pennatisporum MUCC 510 = WAC 13153 * Allocasuarina fraseriana Australia K.M. Taylor EF591976 EF591976 EF591959
Neofusicoccum pistaciae CBS 595.76 Pistacia vera Greece D.G. Zachos KX464163 KX464676 KX464953 KX464008
Neofusicoccum pistaciarum CBS113083 = CPC 5263 * Pistacia vera USA T.J. Michailides KX464186 KX464712 KX464998 KX464027
CBS113084 = CPC 5284 Redwood USA T.J. Michailides KX464187 KX464713 KX464999 KX464028
Neofusicoccum protearum CMW 39280 Acacia karroo Africa KF270041 KF270011
CMW 39282 Acacia karroo Africa KF270043 KF270013
Neofusicoccum ribis CBS 115475 = CMW 7772 * Ribes sp. USA B. Slippers & G. Hudler AY236935 AY236877 AY236906 EU821958
Neofusicoccum sinense CGMCC 3.18315 Unknown wood plant China J.J. Gan KY350148 KY817755 KY350154
Neofusicoccum stellenboschiana CBS 110864 Vitis vinifera South Africa F. Halleen AY343407 AY343348 KX465047 KX464042
Neofusicoccum terminaliae CBS 125263 = CMW26679 * Terminalia sericea South Africa D. Begoude & J. Roux GQ471802 GQ471780 KX465052 KX464045
CBS 125264 = CMW26683 Terminalia sericea South Africa D. Begoude & J. Roux GQ471804 GQ471782 KX465053 KX464046
Neofusicoccum umdonicola CBS 123645 = CMW 14058 * Syzygium cordatum South Africa D. Pavlic EU821904 EU821874 EU821844 EU821934
CBS 123646 = CMW 14060 Syzygium cordatum South Africa D. Pavlic EU821905 EU821875 EU821845 EU821935
Neofusicoccum ursorum CBS 122811 = CMW 24480 * Eucalyptus sp. South Africa H.M. Maleme FJ752746 FJ752709 KX465056 KX464047
CMW 23790 Eucalyptus sp. South Africa H.M. Maleme FJ752745 FJ752708 KX465057
Neofusicoccum viticlavatum CBS 112878 = STE-U 5044 * Vitis vinifera South Africa F. Halleen AY343381 AY343342 KX465058 KX464048
CBS 112977 = STE-U 5041 Vitis vinifera South Africa F. Halleen AY343380 AY343341 KX465059
Neofusicoccum vitifusiforme CBS 110887 = STE-U 5252 * Vitis vinifera South Africa J.M. van Niekerk AY343383 AY343343 KX465061 KX464049
CBS 110880 = STE-U 5050 Vitis vinifera South Africa J.M. van Niekerk AY343382 AY343344
Sphaeropsis chromolaenicola MFLUCC 17-1499 * Chromolaena odorata Thailand A. Mapook MT214366
Sphaeropsis citrigena ICMP 16812 * Citrus sinensis New Zealand S.R. Pennycook, P.R. Johnston & B.C.Paulus EU673328 EU673294 EU673140
Sphaeropsis citrigena (cont.) ICMP 16818 Citrus sinensis New Zealand S.R. Pennycook, P.R. Johnston & B.C.Paulus EU673329 EU673295 EU673141
Sphaeropsis eucalypticola MFLUCC 11-0579 * Eucalyptus sp. Thailand M. Doilom JX646802 JX646867 JX646850
MFLUCC 11-0654 Eucalyptus sp. Thailand M. Doilom JX646803 JX646868 JX646851
Sphaeropsis porosa CBS 110496 = STE-U 5132 * Vitis vinifera South Africa J.M. van Niekerk AY343379 AY343340 EU673130 KX464076
CBS 110574 = STE-U 5046 Vitis vinifera South Africa J.M. van Niekerk AY343378 AY343339
Sphaeropsis ulmicola CBS 174.63 Ulmus glabra Finland MK134681
PB-11f Ulmus glabra Poland MK134682
Sphaeropsis visci CBS 100163 * Vitis vinifera South Africa J.M. van Niekerk EU673324 EU673292 EU673127 KX464077
CBS 186.97 Viscum album Germany T. Graefenhan EU673325 EU673293 EU673128 KX464080

a ALG: Personal culture collection A. Berraf-Tebbal; ATCC: American Type Culture Collection, Virginia, USA; BL: Personal number of B.T. Linaldeddu; BRIP: Queensland Plant Pathology Herbarium, Brisbane, Australia; CAA: Personal culture collection Artur Alves, Universidade de Aveiro, Portugal; CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CERC: Culture collection of China Eucalypt Research Centre, Chinese Academy of Forestry, ZhanJiang, GuangDong, China; CFCC: China Forestry Culture Collection Center, Beijing, China; CGMCC: China General Microbiological Culture Collection Center, Beijing, China; CJA: Collection of J. Abdollahzadeh, Department of Plant Protection, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran; CMM: Culture Collection of Phytopathogenic Fungi ‘Prof. Maria Menezes, Universidade Federal Rural de Pernambuco, Recife, Brazil; CMW: Tree Pathology Co-operative Program, Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; CPC: Working collection of P.W. Crous, housed at CBS; GUCC: Guizhou University Culture Collection; GZCC: Guizhou Academy of Agricultural Sciences Culture Collection, GuiZhou, China; IBL: Personal culture collection, I.B.L. Coutinho; IBP: Personal culture collection, I.B. Prasher; ICMP: International Collection of Microorganisms from Plants, Auckland, New Zealand; IMI: Kew Royal Botanical Gardens, Kew, England; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Iran; JL: Personal culture collection of J. Luque, IRTA, Barcelona, Spain; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; MUCC: Culture collection of Murdoch University, Perth, Australia; PD: Culture Collection, University of California, Davis, USA; STE-U: Culture collection of the Department of Plant Pathology, University of Stellenbosch, South Africa; UCD: University of California, Davis, Plant Pathology Department Culture Collection; UCROK: Department of Plant Pathology and Microbiology, University of California, Riverside; UY: Department of Plant Pathology, University of Minnesota; WAC: Department of Agriculture, Western Australia Plant Pathogen Collection, South Perth, Western Australia; XTBG: Institutional Repository of Xishuangbanna Tropical Botanical Garden.

b ITS, internal transcribed spacer region and intervening 5.8S nrRNA gene; tef1, translation elongation factor 1-alpha; tub2, β-tubulin; rpb2, DNA-directed RNA polymerase II second largest subunit.

* Isolates represent ex-type.

The maximum parsimony (MP) analyses were conducted using PAUP v. 4.0b10 (Swofford 2003), with gaps treated as a fifth character. The characters were unordered and of equal weight with 1 000 random addition replicates. The equally most parsimonious trees were generated using the heuristic search option with the tree bisection-reconnection (TBR) branch swapping. MAXTREES were set to 5 000 and zero-length branches were collapsed. To assess clade stability, a bootstrap analysis was conducted with 1 000 replicates. Tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) were recorded to evaluate the trees (Hillis & Bull 1993).

The maximum-likelihood (ML) analyses for each dataset were conducted using PhyML v. 3.0 (Guindon et al. 2010). The software package jModeltest v. 2.1.5 (Darriba et al. 2012) was used to determine the best nucleotide substitution model for each dataset. In PhyML, the retention of the maximum number of 1 000 trees was set and nodal support was determined by non-parametric bootstrapping with 1 000 replicates. For both the MP and ML analyses, the phylogenetic trees were viewed in MEGA v. 7.0.26 and FigTree v. 1.4.4 (http://tree.bio.ed.ac. uk/software/figtree).

Morphology

Representative isolates of Botryosphaeriaceae that were identified as new species based on DNA sequence analysis were selected for morphological study. Sporulation was induced on pine needle agar (PNA) (Smith et al. 1996) by incubating cultures at 25 °C in 12/12 h fluorescent light/dark cycle for 4–6 wk. Sporocarps were embedded in a Leica Biosystem Tissue Freezing Medium (Leica Biosystems Nussloch GmbH, Nussloch, Germany) and sectioned (8 μm thick) using a freezing microtome (CryoStar NX50 HOP, Thermo Fisher Scientific, Walldorf, Germany) at -20 °C (Chen et al. 2018). Conidia and other microstructures were examined with a compound microscope (Eclipse 80i, Nikon, Japan) and images were recorded with a Nikon digital camera (NIS-Elements F3.0, Nikon, Japan). Measurements were made with Fiji-ImageJ software (Schindelin et al. 2012). One hundred conidia were measured per isolate, and 30 measurements were taken of other morphological structures. Results are presented as (minimum–) (mean – standard deviation) – (mean + standard deviation) (–maximum). The average length/average width ratio (L/W) of the conidial measurements were also calculated.

Colony characters on PDA were noted and colony colours were determined according to the colour charts of Rayner (1970). To determine growth rates in culture, agar plugs (5 mm diam) were taken from the edge of actively growing cultures of each representative isolate and transferred onto the centre of 90 mm diam Petri dishes containing PDA. Cultures were incubated at five temperature intervals from 5–40 °C in the dark. Five replicate plates of each representative isolate were incubated at each temperature. Perpendicular colony diameters were measured daily until the fastest growing cultures reached the edge of the Petri dish.

Pathogenicity tests

At least two representative isolates from each identified group, except for those with only one isolate, were selected for pathogenicity testing in this study. Inoculation tests were conducted both in vitro and in vivo. For in vitro inoculation, isolates were used to inoculate detached healthy green shoots (40 cm long, 0.6–1 cm diam) collected from Citrus reticulata trees and 10 shoots were inoculated with each isolate. One wound per shoot was made using a cork borer (5 mm diam) and a mycelial plug taken from the margins of colonies grown on PDA for 5 d in the dark was placed on the freshly wounded surface of each shoot, and the inoculated area was covered with Parafilm. The control treatment was inoculated with sterile PDA plugs. The inoculated shoots and controls were covered with liquid paraffin at their ends to prevent desiccation and incubated at 25 °C in moist chambers. Eight days after inoculation, the disease incidences were calculated and the internal lesions or wound lengths were measured. Data were analysed by one-way analysis of variance (ANOVA) using SPSS Statistics 20 software (SPSS 2011). To prove Koch’s postulates, fungi were re-isolated by cutting small pieces of necrotic tissue from the edges of each lesion and plating them in PDA plates at 25 °C. The species were confirmed based on morphology.

For in vivo inoculation, the pathogenicity test was conducted on 6-yr-old healthy plants of Cocktail grapefruit (C. paradisi × C. reticulata). The plants were grown in vinyl house of the Xielong Family Farm in Kecheng District, Quzhou City, Zhejiang Province from 24 June to 9 July 2021. During this time, the environmental temperature ranged from 20–38 °C. Each representative isolate, as well as the control, was inoculated onto 10 shoots. After 15 d, the symptoms and disease incidences were assessed. Re-isolation was also conducted in the same way to fulfil Koch’s postulates.

RESULTS

Isolates

A total of 111 isolates from 88 collected citrus samples exhibited typical morphological characteristics of Botryosphaeriaceae. Eighty-one isolates were collected from Zhejiang, seven from Guangxi, six from Guangdong, four respectively from Chongqing, Fujian and Shaanxi, two respectively from Hunan and Jiangxi, and one from Shanghai. Among them, 52 isolates were obtained from twigs and branches with dieback, 31 were associated with branches and trunks with canker, and 28 from gummosis symptoms. In terms of Citrus species, 42 isolates were obtained from C. unshiu, 25 from C. reticulata, 10 from C. sinensis, nine from C. maxima, one from C. limon, and 24 from hybrids.

Phylogenetic analyses

The ITS and tef1 sequences were amplified for all 111 isolates obtained in this study, and blast results indicated that these isolates resided in Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia, Neodeightonia, Neofusicoccum and Sphaeropsis. Fifty-seven representative isolates were subsequently selected to be sequenced for their tub2 and rpb2 loci (Table 1). Datasets for the seven genera, the parameters of the statistical values of the trees for the MP analyses and the best-fit substitution models for ML analyses are provided in Table 3. All sequences of Botryosphaeriaceae species obtained in this study were deposited in GenBank (Table 1).

Table 3.

Datasets used and statistics resulting from phylogenetic analyses in the current study.

Genus Dataset Maximum likelihood

Subst. model1 NST2 Rate matrix Ti/Tv ratio3 p-inv Gamma Rates
Botryosphaeria ITS TrN+I 6 1.0000 1.4653 1.0000 1.0000 7.9294 0.8090 equal
tef1 TPM3uf+I 6 0.3783 2.6378 1.0000 0.3783 2.6378 0.6000 equal
tub2 TrN+I 6 1.0000 6.0784 1.0000 1.0000 14.3581 0.7170 equal
rpb2 TIM3+G 6 9756.0724 15799.8965 1.0000 9756.0724 70110.9963 0.1480 gamma
ITS/tef1/tub2 TrN+I 6 1.0000 3.4860 1.0000 1.0000 7.2459 0.761 equal
Diplodia ITS TVMef+I+G 6 6.1639 23.8085 4.6946 13.6614 23.8085 0.6580 0.6850 gamma
tef1 TrN+G 6 1.0000 3.3131 1.0000 1.0000 5.5220 0.5530 gamma
tub2 TrN+I 6 1.0000 3.1072 1.0000 1.0000 5.7192 0.676 equal
rpb2 TrN+G 6 1.0000 5.1679 1.0000 1.0000 13.7446 0.228 gamma
ITS/tef1/tub2 TrN+I+G 6 1.0000 3.6215 1.0000 1.0000 5.0395 0.5940 1.3320 gamma
Dothiorella ITS TrN+I+G 6 1.0000 1.4234 1.0000 1.0000 3.2819 0.4180 0.6840 gamma
tef1 TPM2uf+G 6 2.0341 5.0971 2.0341 1.0000 5.0971 0.765 gamma
tub2 HKY+I+G 2 1.7295 0.5600 0.9450 gamma
rpb2 TrN+I 6 1.0000 3.8616 1.0000 1.0000 10.6645 0.6030 equal
ITS/tef1/tub2 TIM2+G 6 1.2403 2.7176 1.2403 1.0000 4.1029 0.2160 gamma
Lasiodiplodia ITS K80+I 2 2.4444 0.7840 equal
tef1 K80+G 2 1.7723 0.4130 gamma
tub2 TrNef+I 6 1.0000 1.6752 1.0000 1.0000 6.7164 0.6540 equal
rpb2 TrNef+G 6 1.0000 5.3397 1.0000 1.0000 12.4894 0.3110 gamma
ITS/tef1/tub2/rpb2 TrN+I+G 6 1.0000 3.8560 1.0000 1.0000 7.6054 0.5410 0.6090 gamma
Neodeightonia ITS TPM3uf+I+G 6 3.2767 4.6927 1.0000 3.2767 4.6927 0.6640 0.5000 gamma
tef1 TIM1 6 1.0000 1.0118 0.2049 0.2049 2.8685 equal
tub2 TIM1+I 6 1.0000 1.2183 0.3558 0.3558 2.4674 0.6860 equal
ITS/tef1/tub2 TIM1+G 6 1.0000 1.2491 0.5067 0.5067 3.6475 0.1940 gamma
Neofusicoccum ITS TIM1ef+I+G 6 1.0000 6.4363 2.1879 2.1879 17.3039 0.5780 0.6010 gamma
tef1 HKY+G 2 2.5159 0.7750 gamma
tub2 TrN+G 6 1.0000 3.1372 1.0000 1.0000 6.7052 0.2270 gamma
rpb2 TrN+G 6 1.0000 5.5358 1.0000 1.0000 17.053 0.235 gamma
ITS/tef1/tub2/rpb2 K80+G 2 2.265 gamma
Sphaeropsis ITS HKY+I+G 2 1.7294 0.5610 0.9480 gamma
tef1 HKY+I+G 2 1.7294 0.5610 0.9480 gamma
tub2 TIM2+G 6 1.2403 2.7176 1.2403 1.0000 4.1029 0.2160 gamma
rpb2 TIM2+G 6 1.2403 2.7176 1.2403 1.0000 4.1029 0.2160 gamma
ITS/tef1/tub2/rpb2 HKY+I+G 2 1.7294 0.5610 gamma
Botryosphaeria ITS 26 504 25 1 39 0.7949 0.8769 0.6970 0.2051
tef1 26 347 103 42 129 0.8992 0.9378 0.8433 0.1008
tub2 21 406 16 1 21 0.9048 0.9444 0.8545 0.0952
rpb2 14 659 12 3 18 0.8889 0.9583 0.8519 0.1111
ITS/tef1/tub 26 1257 144 3 193 0.8601 0.9129 0.7852 0.1399
Diplodia ITS 47 535 91 1354 233 0.6652 0.8534 0.5677 0.3348
tef1 47 287 117 5000 275 0.6291 0.9016 0.5672 0.3709
tub 39 388 46 1103 95 0.6105 0.8571 0.5233 0.3895
rpb2 7 594 41 2 61 0.8033 0.7857 0.6311 0.1967
ITS/tef1/tub2 47 1210 42 42 658 0.5881 0.8536 0.5020 0.4119
Dothiorella ITS 63 496 97 384 287 0.5714 0.8955 0.5117 0.4286
tef1 63 324 217 400 791 0.5310 0.8594 0.4563 0.4690
tub2 45 387 95 185 203 0.6946 0.8758 0.6083 0.3054
rpb2 16 594 83 2 145 0.6621 0.8333 0.5517 0.3379
ITS/tef1/tub2 63 1540 409 6 1336 0.5427 0.8584 0.4658 0.4573
Lasiodiplodia ITS 99 486 49 5000 89 0.6517 0.8905 0.5803 0.3483
tef1 98 291 132 802 284 0.6092 0.8911 0.5428 0.3908
tub2 90 345 34 10 54 0.7222 0.9227 0.6664 0.2778
rpb2 77 521 86 201 166 0.6024 0.8981 0.5411 0.3976
ITS/tef1/tub2/rpb2 99 1643 313 670 956 0.485356 0.8422 0.4087 0.5146
Neodeightonia ITS 15 502 51 1 111 0.7840 0.8490 0.6650 0.2160
tef1 9 229 14 1 19 0.8420 0.8750 0.7370 0.1580
tub2 7 418 6 1 9 0.8890 0.8570 0.7620 0.1110
ITS/tef1/tub2 15 1150 78 3 162 0.7346 0.8114 0.5960 0.2654
Neofusicoccum ITS 57 494 54 28 146 0.5548 0.8516 0.4725 0.4452
tef1 55 280 115 5000 273 0.6923 0.9012 0.6239 0.3077
tub2 51 422 67 5000 130 0.6231 0.8345 0.5199 0.3769
rpb2 33 560 75 68 143 0.6364 0.8267 0.5261 0.3636
ITS/tef1/tub2/rpb2 57 1756 334 24 817 0.5704 0.8208 0.4682 0.4296
Sphaeropsis ITS 10 518 54 1 19 1.0000 1.0000 1.0000 1.0000
tef1 10 297 77 1 117 0.8462 0.8583 0.7262 0.1538
tub2 9 375 20 3 21 0.8095 0.8261 0.6687 0.1905
rpb2 5 557 18 2 27 0.7410 0.6110 0.4530 0.2590
ITS/tef1/tub2/rpb2 10 1747 130 2 188 0.8245 0.8245 0.6797 0.1755

1 Subst. model = best fit substitution model.

2 NST = number of substitution rate categories.

3 Ti/Tv ratio = transition/transversion ratio.

4 bp = base pairs.

5 PIC = number of parsimony informative characters.

6 CI = consistency index.

7 RI = retention index.

8 RC = rescaled consistency index.

9 HI = homoplasy index.

Species of Botryosphaeria

Isolates clustered into two phylogenetic groups (Group A and B) for the individual genes (ITS, tef1, tub2, rpb2), as well as the combined gene dataset (Fig. 2, S1a–d). For the ITS sequences, isolates BE3, BE78, BE85 and BE86 (Group A) grouped with several species, while isolates BE1, BE2, BE63 and BE66 (Group B) formed a clade distinct from the other species (Fig. S1a). For the tef1 and rpb2 and combined ITS/tef1/tub2/rpb2 datasets, isolates in Group A were closely related to B. fabicerciana, and isolates in Group B were most closely related to B. dothidea (Fig. 2, S1b, d). Therefore, isolates in Group A were identified as B. fabicerciana, and isolates in Group B were identified as B. dothidea.

Fig. 2.

Fig. 2

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1 and tub2 sequence alignments of Botryosphaeria. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Neofusicoccum parvum (CMW 9081).

Species of Diplodia

Isolate BE4 (Group C) clustered more closely to D. seriata and D. galiicola in the ITS datasets (Fig. S2a), while more closely to D. seriata and D. sapinea in the tef1 datasets (Fig. S2b). For the tub2 and rpb2 sequences, isolate BE4 clustered with D. seriata (Fig. S2c–d). The analyses of the combined ITS, tef1, tub2 and rpb2 sequences demonstrated that isolate BE4 was most closely related to D. seriata (Fig. 3).

Fig. 3.

Fig. 3

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1 and tub2 sequence alignments of Diplodia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Lasiodiplodia theobromae (CBS 164. 96).

Species of Dothiorella

Eight isolates clustered in three clades (Group D–F). Isolate BE17 in Group D grouped with Do. alpina and Do. magnoliae based on the ITS sequences (Fig. S3a). For the tef1 sequences, isolate BE17 formed an independent lineage close to Do. alpina and Do. acericola (Fig. S3b). For the tub2 and rpb2 sequences, isolate BE17 formed an independent lineage (Fig. S3c, d). For the combined ITS, tef1 and tub2 sequences, isolate BE17 clustered with Do. alpina (Fig. 4). For Group E, the sequence analyses of ITS, tef1, tub2 and ITS/tef1/tub2 sequences showed that isolates BE16 and BE74 clustered in the same clade (ITS, tub2) or close (tef1, ITS/tef1/tub2) to Do. plurivora (rpb2 sequences are not available for Do. plurivora) (Fig. 4, S3a–c). Thus, isolates in Group D were identified as Do. alpina, while those in Group E were identified as Do. plurivora.

Fig. 4.

Fig. 4

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1 and tub2 sequence alignments of Dothiorella. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Neofusicoccum parva (CMW 9081).

Isolates in Group F (BE5, BE7, BE8, BE9, BE71) clustered close to Do. striata based on the ITS and tef1 datasets (Fig. S3a–b), but formed independent clades that were separated from Do. striata with high bootstrap values in the tub2, rpb2 and combined ITS/tef1/tub2 datasets (tub2, ML/MP = 92 % / 94 %; rpb2, ML/MP = 100 % / 100 %; ITS/tef1/tub2, ML/MP = 84 % / 97 %) (Fig. 4, S3c–d). Thus, isolates in Group F were considered as an undescribed species in Dothiorella.

Species of Lasiodiplodia

Isolates resided in nine groups (Group G–O) based on the tef1 and combined ITS/tef1/tub2/rpb2 datasets (Fig. 5, S4b). Isolates in Group G (BE27, BE30, BE36, BE41, BE42, BE100) were closest to L. iraniensis and various other species based on the ITS, tub2 and rpb2 datasets (Fig. S4a, c–d). For the tef1 and combined ITS/tef1/tub2/rpb2 sequences, the six isolates were closest to L. iraniensis (Fig. 5, S4b). Thus, the six isolates in Group G were identified as L. iraniensis.

Fig. 5.

Fig. 5

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1, tub2 and rpb2 sequence alignments of Lasiodiplodia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

For isolates in Group H (BE20, BE21, BE25) and Group J (BE32, BE80, BE87, BE88), the analyses of the ITS, tef1, tub2 and rpb2 sequences indicated that isolates in Group H clustered into the same (ITS, tub2, rpb2) clade or close (tef1) to L. theobromae, while isolates in Group J clustered into the same (rpb2) clade or close (ITS, tef1) to L. microconidia (Fig. S4a–d). The combined ITS/tef1/tub2/rpb2 datasets showed that isolates in Group H were more closely related to L. theobromae, while isolates in Group J clustered with L. microconidia (Fig. 5).

Isolates in Group I (BE28, BE34, BE40, BE51) and Group L (BE31, BE59) clustered with various Lasiodiplodia species based on ITS, tub2 and rpb2 datasets (Fig. S4a, c–d), but formed independent clades based on the tef1 and combined ITS/tef1/tub2/rpb2 trees, with high bootstrap values (Group I: tef1, ML/MP = 99 % / 100 %; ITS/tef1/tub2/rpb2, ML/MP = 99 % / 100 %; Group L: tef1, ML/MP = 98 % / 99 %; ITS/tef1/tub2/rpb2, ML/MP = 99 % / 100 %) (Fig. 5, S4b). Therefore, isolates in Group I and Group L were considered to represent two novel species.

Isolates in Group K (BE10, BE11, BE12, BE19, BE26, BE37) clustered with L. pseudotheobromae and various other species based on the ITS and tub2 datasets (Fig. S4a, c). For the analyses of tef1, rpb2 and the combined ITS/tef1/tub2/rpb2 datasets, the six isolates resided in the same clade with L. pseudotheobromae (Fig. 5, S4b, d). Therefore, the isolates in Group K were treated as L. pseudotheobromae. Isolates in Group M (BE13, BE38, BE45, BE89, BE102, BE104) clustered into the same (ITS, rpb2) clade or close (tef1, rpb2) to L. citricola (Fig. S4a–d). The combined ITS/tef1/tub2/rpb2 datasets showed that isolates in Group M clustered with L. citricola (Fig. 5).

Isolate BE44 in Group N resided in a clade with L. citricola based on the analyses of the ITS and tub2 datasets (Fig. S4a, c). For the tef1 datasets, BE44 formed an independent lineage phylogenetically close to L. aquilariae (Fig. S4b). For the rpb2 datasets, BE44 grouped with various other species (Fig. S4d). The analyses of the combined ITS/tef1/tub2/rpb2 datasets indicated that isolate BE44 formed an independent lineage that was distinguished from other known phylogenetically related species (Fig. 5). Isolates in Group O (BE33, BE50) grouped together with various other Lasiodiplodia species in the ITS, tub2 and rpb2 trees (Fig. S4a, c–d). For the tef1 datasets, the two isolates formed two independent clades but with low bootstrap support based on the tef1 in the ML analyses, while they clustered together with Group L in the MP analyses (data not shown). The analyses of the combined ITS/tef1/tub2/rpb2 datasets indicated that isolates in Group O formed an independent clade with high support bootstrap values (ML/MP = 74 % / 88 %) (Fig. 5). Consequently, isolates in Group N and Group O were identified as two new species of Lasiodiplodia.

Species of Neodeightonia

Isolate BE14 (Group P) grouped with N. subglobosa in one clade with high support value on the basis of the phylogenetic analyses for the ITS, tef1, tub2 and ITS/tef1/tub2 datasets (Fig. 6, S5a–d).

Fig. 6.

Fig. 6

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1 and tub2 sequence alignments of Neodeightonia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

Species of Neofusicoccum

Phylogenetic analyses of ITS and tef1 consistently indicated that isolate BE15 (Group Q) resided in one phylogenetic clade with Ne. parvum (Fig. S6a–b), and with Ne. pennatisporum based on tub2 (Fig. S6c). Isolate BE15 clustered with Ne. parvum, Ne. cryptoaustrale and Ne. mangiferae based on rpb2 (Fig. S6d). The phylogeny based on the combined ITS/tef1/tub2/rpb2 sequences indicated that isolate BE15 was closely related to Ne. parvum (Fig. 7).

Fig. 7.

Fig. 7

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1, tub2 and rpb2 sequence alignments of Neofusicoccum. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Dothiorella viticola (CBS 117009).

Species of Sphaeropsis

Isolate BE18 (Group R) formed an independent lineage that was distinct from any known species of Sphaeropsis based on the phylogenetic analyses for ITS, tef1, tub2, rpb2 and the combined four gene datasets. The bootstrap values associated with the other species were higher than 50 % in ITS, tef1, rpb2 and the combined datasets (ITS, ML/MP = 68 % / 64 %; tef1, MP = 93 %; rpb2, MP = 100 %; ITS/tef1/tub2/rpb2, ML/MP = 50 % / 55 %) (Fig. 8, S7a–b, d). Therefore, isolate BE18 was treated as a novel species of Sphaeropsis.

Fig. 8.

Fig. 8

Phylogenetic tree generated by maximum likelihood analyses based on the combined ITS, tef1, tub2 and rpb2 sequence alignments of Sphaeropsis. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

Morphology and taxonomy

The selected isolates for morphological studies produced pycnidia on PNA within 4–6 wk. No sexual structures were observed in this study. Based on DNA sequences and morphology, 18 species belonging to seven genera were identified. Of these, Botryosphaeria dothidea, B. fabicerciana, Diplodia seriata, Dothiorella alpina, Do. plurivora, Lasiodiplodia citricola, L. iraniensis, L. microconidia, L. pseudotheobromae, L. theobromae, Neodeightonia subglobosa and Neofusicoccum parvum are known species. The remaining six species are described below.

Dothiorella citrimurcotticola X.E. Xiao, P.W. Crous & H.Y. Li, sp. nov. — MycoBank MB 840681; Fig. 9

Fig. 9.

Fig. 9

Dothiorella citrimurcotticola. a. Conidioma formed on PNA; b. section view of conidioma; c–d. conidiogenous cells and developing conidia; e–f. conidia; g. colony growing on PDA after 3 d. — Scale bars: a = 100 μm; b = 50 μm; c–f = 10 μm; g = 2.1 cm.

Etymology. Referring to the citrus host (Murcott) which it was isolated.

Typus. CHINA, Chongqing Municipality, Wanzhou City, from a twig of Murcott (C. reticulata × C. sinensis), 23 Mar. 2019, H.Y. Li & X.E. Xiao, conidiomata induced on PNA (holotype ZJUE H-0008, culture ex-type CGMCC 3.20394 = BE8).

Sexual morph unknown. Conidiomata pycnidial, produced on PNA within 2–4 wk, dark brown to black, up to 823 μm diam, globose, superficial or semi-immersed, unilocular, thick-walled. Conidiophores absent. Conidiogenous cells cylindrical to fusiform or lageniform, hyaline, thin-walled, smooth, 4.5–10.5 × 2–5 μm. Conidia subcylindrical to ellipsoid or ovoid, initially hyaline, thin-walled, aseptate, becoming brown, thick-walled, 1-septate, externally smooth, internally verrucose, apex rounded, base truncate or rounded, (21.5–)23–25.5(–27) × (8.5–)9.5–11(–14) μm (av. = 24.4 × 10.3 μm, n = 100; L/W ratio = 2.4) (Table 4).

Table 4.

Conidial measurements of Botryosphaeriaceae species.

Species1 Conidia
Paraphyses
Reference
Conidial size (μm) (L × W)2 Mean (μm) (L × W)3 L/W4 long (μm)5 wide (μm)6
Do. citrimurcotticola (21.5–)23–25.5(–27)× (8.5–)9.5–11(–14) 24.4×10.3 2.4 This study
Do. striata (21–)23–26(–29.4) × (8.9–)9–12(–15.1) 25.1 × 10.7 2.4 Abdollahzadeh et al. (2014)
Do. uruguayensis (17–)22–22.5(–26.5) × (7–)9–9.5(–12) 22 × 9.25 2.4 Pérez et al. (2010)
Lasiodiplodia acaciae (21.5–)25–29.5(–31) × (11–)12–14(–15) 27.3 × 12.9 2.1 69 2–5 Zhang et al. (2021)
L. aquilariae (23–)25–28(–29) × 12–16 26.9 × 14.1 1.8 100 3 Wang et al. (2019)
L. cinnamomi (17.5–)18.7–21.1(–22.4) × (11.5–)12.7–14.1(–15.5) 19.9 × 13.4 1.5 106 3–4 Jiang et al. (2018)
L. citricola (20–)22–27(–31) × (10.9–)12–17(–19) 24.5 × 15.4 1.6 125 3–4 Abdollahzadeh et al. (2010)
L. guilinensis (23–)28–31(–33.5)× (13.5–)15–16.5(–17) 29.6 ×15.7 1.9 75 2–5 This study
L. huangyanensis (21–)28–32.5(–34) × (13–)14–16(–17) 30.1× 15 2 82 3–4 This study
L. linhaiensis (24.5–)27–30(–32)× (12.5–)13.5–15(–16) 28.5 × 14.2 2 80 2–6 This study
L. microconidia (18–)19–22(–23) × 10–15 20.8 × 13.2 1.5 90 3 Wang et al. (2019)
L. ponkanicola (16–)23.5–27.5(–28.5)× (11–)13–14.5(–15.5) 25.4 × 13.7 1.9 87 2–5 This study
Sphaeropsis citrigena (27–)28–33(–34) × (14.5–)15–18.5(–19) 30.5 × 16.8 1.8 25 3–5 Phillips et al. (2008)
S. linhaiensis (26.5–)28.5–35(–38)× (11.5–)14–18(–19.5) 31.6 × 15.9 2 27 1–5 This study

1 Isolates and measurements in bold were examined in this study.

2 Minimum – (average – standard deviation) – (average + standard deviation) – maximum or minimum – maximum, L × W = length × width.

3 L × W = average length × average width.

4 L/W = average length/average width.

5 Maximum.

6 Maximum or minimum – maximum.

Culture characteristics — Colonies on PDA have abundant aerial mycelia, initially leaden grey in the centre, becoming pale mouse grey at the surface and greenish olivaceous to dull green at the reverse. Colonies cover the 90 mm plates after 3 d at the optimum temperature of 25 °C. No growth was observed at 40 °C. After 3 d, colonies at 5 °C, 10 °C, 15 °C, 20 °C, 30 °C and 35 °C reach 11 mm, 11 mm, 59 mm, 79 mm, 42 mm and 12 mm, respectively.

Additional materials examined. CHINA, Zhejiang Province, Yongquan Town, from a twig of C. unshiu, Aug. 2018, H.Y. Li, conidiomata induced on PNA (holotype ZJUE H-0005, culture ex-type CGMCC 3.20392 = BE5); Chongqing Municipality, Wanzhou City, from a twig of Murcott (C. reticulata × C. sinensis), 23 Mar. 2019, H.Y. Li & X.E. Xiao, conidiomata induced on PNA (ZJUE H-0009, culture CGMCC 3.20395 = BE9); Zhejiang Province, Quzhou City, from a twig of C. maxima, 27 Apr. 2019, H.Y. Li, conidiomata induced on PNA (ZJUE H-0007, culture CGMCC 3.20393 = BE7).

Notes — Phylogenetically, Do. citrimurcotticola is closely re-lated to Do. striata and Do. uruguayensis, but morphologically it can be distinguished based on their average conidial dimensions. Conidia of Do. citrimurcotticola (av. 24.4 × 10.3; L/W = 2.4) are larger than Do. uruguayensis (av. 22 × 9.25; L/W = 2.4) (Pérez et al. 2010) but smaller than Do. striata (av. 25.1 × 10.7; L/W = 2.4) (Abdollahzadeh et al. 2014) (Table 4). Moreover, Do. citrimurcotticola differs from these species based on nucleotide differences in ITS (Do. striata: 5 bp, Do. uruguayensis: 1 bp), tef1 (Do. striata: 5 bp, Do. uruguayensis: 15 bp and including three gaps), tub2 (Do. striata: 4 bp, Do. uruguayensis: 6 bp) and rpb2 loci (Do. striata: 8 bp).

Lasiodiplodia guilinensis X.E. Xiao, P.W. Crous & H.Y. Li, sp. nov. — MycoBank MB 840682; Fig. 10

Fig. 10.

Fig. 10

Lasiodiplodia guilinensis. a–b. Pale yellow to saffron yellow conidial mass released from conidiomata formed on PNA; c. section view of conidioma; d. paraphyses; e–f. conidia developing on conidiogenous cells between paraphyses; g. hyaline, aseptate conidia; h–i. dark-brown, 1-septate conidia at two different focal planes to show the longitudinal striations; j. colony growing on PDA after 2 d. — Scale bars: a–b = 200 μm; c = 50 μm; d–i = 10 μm; j = 2 cm.

Etymology. Referring to the city, Guilin, where it was collected.

Typus. CHINA, Guangxi Province, Guilin City, from a twig of C. sinensis cv. Valencia, 26 Mar. 2019, H.Y. Li & X.E. Xiao, conidiomata induced on PNA (holotype ZJUE H-0031, culture ex-type CGMCC 3.20378 = BE31).

Sexual morph unknown. Conidiomata stromatic, produced on PNA within 2–4 wk, superficial or semi-immersed, dark brown to black, up to 2 mm diam, solitary or aggregated, unilocular, covered by dense mycelium, globose, thick-walled, often releasing pale yellow to saffron yellow conidial tendrils or mass. Paraphyses hyaline, cylindrical, septate, unbranched, ends rounded, up to 75 μm long, 2–5 μm wide, formed among conidiogenous cells. Conidiophores absent. Conidiogenous cells holoblastic, hyaline, smooth, thin-walled, cylindrical, 8–54 × 3–9 μm. Conidia initially hyaline, aseptate, ellipsoid to ovoid, thin-walled with granular content, rounded at apex, base round or truncate, becoming dark brown, 1-septate with longitudinal striations, (23–)28–31(–33.5) × (13.5–)15–16.5(–17) μm (av. = 29.6 × 15.7 μm, n = 100; L/W ratio = 1.9) (Table 4).

Culture characteristics — Colonies on PDA with moderately dense aerial mycelium, initially white to smoke grey, turning grey olivaceous on the surface and greenish grey in reverse, becoming dark slate-blue with age. Colonies cover the 90 mm plates after 2 d in the dark at the optimum temperature of 25–30 °C. No growth was observed at 5 °C. After 2 d, colonies at 10 °C, 15 °C, 20 °C, 35 °C and 40 °C reach 11 mm, 32 mm, 64 mm, 72 mm and 12 mm, respectively.

Additional material examined. CHINA, Zhejiang Province, Yongquan Town, from the branch of C. unshiu, 26 Sept. 2017, H.Y. Li, conidiomata induced on PNA (ZJUE H-0059, culture CGMCC 3.20379 = BE59).

Notes — Phylogenetically, L. guilinensis is closely related to L. aquilariae and L. citricola, but morphologically it can be separated from these species based on average conidial dimensions and length of its paraphyses. Conidia of L. guilinensis (av. 29.6 × 15.7; L/W = 1.9) are larger than those of L. aquilariae (av. 26.9 × 14.1; L/W = 1.8) (Wang et al. 2019) and L. citricola (av. 24.5 × 15.4; L/W = 1.6) (Abdollahzadeh et al. 2010). In terms of paraphyses, those of L. guilinensis (up to 75 μm long) are shorter than L. aquilariae (up to 100 μm long) (Wang et al. 2019) and L. citricola (up to 125 μm long) (Abdollahzadeh et al. 2010) (Table 4). Furthermore, L. guilinensis differs from these species by nucleotide differences in ITS (L. aquilariae: 6 bp, L. citricola: 3 bp), tef1 (L. aquilariae: 16 bp, L. citricola: 14 bp), tub2 (L. citricola: 3 bp) and rpb2 loci (L. aquilariae: 3 bp).

Lasiodiplodia huangyanensis X.E. Xiao, P.W. Crous & H.Y. Li, sp. nov. — MycoBank MB 840683; Fig. 11

Fig. 11.

Fig. 11

Lasiodiplodia huangyanensis. a. Conidioma formed on PNA; b–c. section view of conidiomata; d–g. conidia developing on conidiogenous cells between paraphyses; h. hyaline, aseptate conidia; i–j. dark-brown, 1-septate conidia at two different focal planes to show the longitudinal striations; k. colony growing on PDA after 2 d. — Scale bars: a = 200 μm; b–c = 50 μm; d–j = 10 μm; k = 2.1 cm.

Etymology. Referring to the district, Huangyan, where it was collected.

Typus. CHINA, Zhejiang Province, Huangyan District, from a twig of C. reti-culata cv. Succosa, 22 Jan. 2019, X.E. Xiao & Q.B. Huang, conidiomata induced on PNA (holotype ZJUE H-0033, culture ex-type CGMCC 3.20380 = BE33).

Sexual morph unknown. Conidiomata stromatic, formed on PNA within 2–4 wk, superficial or semi-immersed, dark brown to black, up to 1.5 mm diam, solitary or aggregated, unilocular, covered by dense mycelium, globose, thick-walled, often releasing in pale yellow to saffron yellow conidial tendrils or mass. Paraphyses hyaline, cylindrical, septate, unbranched, ends rounded, up to 82 μm long, 3–4 μm wide, formed among conidiogenous cells. Conidiophores absent. Conidiogenous cells holoblastic, hyaline, smooth, thin-walled, cylindrical, 8–35 × 3.5–7 μm. Conidia initially hyaline, aseptate, ellipsoid to ovoid, thin-walled with granular content, rounded at apex, base round or truncate, becoming dark brown, 1-septate with longitudinal striations, (21–)28–32.5(–34) × (13–)14–16(–17) μm (av. = 30.1 × 15 μm, n = 100; L/W ratio = 2) (Table 4).

Culture characteristics — Colonies on PDA with moderately dense aerial mycelium, initially white to smoke grey, turning grey olivaceous on the surface and greenish grey in reverse, becoming dark slate-blue with age. Colonies cover the 90 mm plates after 2 d in the dark at the optimum temperature of 25–30 °C. No growth was observed at 5 °C. After 2 d, colonies at 10 °C, 15 °C, 20 °C, 35 °C and 40 °C reach 10 mm, 24 mm, 53 mm, 28 mm and 12 mm, respectively.

Additional material examined. CHINA, Zhejiang Province, Linhai City, from the branch of C. unshiu, 13 Dec. 2018, W.L. Li, conidiomata induced on PNA (ZJUE H-0050, culture CGMCC 3.20381 = BE50).

Notes — Phylogenetically, L. huangyanensis is closely related to L. cinnamomi and L. ponkanicola. Morphologically, however, it can be distinguished based on the average conidial dimensions and length of its paraphyses. Conidia of L. huangyanensis (av. 30.1 × 15; L/W = 2) are larger than L. cinnamomi (av. 19.9 × 13.4; L/W = 1.5) (Jiang et al. 2018) and L. ponkanicola (av. 25.4 × 13.7; L/W = 1.9) (this study). Moreover, the paraphyses of L. huangyanensis (up to 82 μm long) are shorter than those of L. cinnamomi (up to 106 μm long) (Jiang et al. 2018) and L. ponkanicola (up to 87 μm long) (this study) (Table 4). Furthermore, L. huangyanensis differs from these species by nucleotide differences in ITS (L. ponkanicola: 3 bp), tef1 (L. cinnamomi: 7 bp, L. ponkanicola: 10 bp), tub2 (L. ponkanicola: 3 bp) and rpb2 loci (L. cinnamomi: 9 bp).

Lasiodiplodia linhaiensis X.E. Xiao, P.W. Crous & H.Y. Li, sp. nov. — MycoBank MB 840684; Fig. 12

Fig. 12.

Fig. 12

Lasiodiplodia linhaiensis. a. Pale yellow conidial mass released from conidioma formed on PNA; b. section view of conidioma; c–e. conidia developing on conidiogenous; f. hyaline, aseptate conidia; g–h. dark-brown, 1-septate conidia at two different focal planes to show the longitudinal striations; i. colony growing on PDA after 2 d. — Scale bars: a = 200 μm; b = 50 μm; c–h = 10 μm; i = 1.8 cm.

Etymology. Referring to the city, Linhai, where it was collected.

Typus. CHINA, Zhejiang Province, Linhai City, from the branch of C. unshiu, 14 Dec. 2018, W.L. Li, conidiomata induced on PNA (holotype ZJUE H-0051, culture ex-type CGMCC 3.20386 = BE51).

Sexual morph unknown. Conidiomata stromatic, produced on PNA within 2–4 wk, superficial or semi-immersed, dark brown to black, up to 950 μm diam, solitary or aggregated, unilocular, covered by dense mycelium, globose, thick-walled, often releasing in pale-yellow to saffron-yellow conidial tendrils or mass. Paraphyses hyaline, cylindrical, septate, unbranched, ends rounded, up to 80 μm long, 2–6 μm wide, formed among conidiogenous cells. Conidiophores absent. Conidiogenous cells holoblastic, hyaline, smooth, thin-walled, cylindrical, 7.5–22.5 × 3–5.5 μm. Conidia initially hyaline, aseptate, ellipsoid to ovoid, thin-walled with granular content, rounded at apex, base round or truncate, becoming dark brown, 1-septate with longitudinal striations, (24.5–)27–30(–32) × (12.5–)13.5–15(–16) μm (av. = 28.5 × 14.2 μm, n = 100; L/W ratio = 2) (Table 4).

Culture characteristics — Colonies on PDA with slightly dense aerial mycelium, initially white to smoke grey, turning grey olivaceous on the surface and greenish grey in reverse, becoming dark slate-blue with age. Colonies cover the 90 mm plates after 2 d in the dark at the optimum temperature of 25–30 °C. No growth was observed at 5 °C. After 2 d, colonies at 10 °C, 15 °C, 20 °C, 35 °C and 40 °C reach 12 mm, 18 mm, 42 mm, 22 mm and 11 mm, respectively. Isolates produced a pink pigment in PDA cultures at 35 °C.

Additional materials examined. CHINA, Guangxi Province, Guilin City, from the dieback of C. sinensis cv. Valencia, 26 Mar. 2019, H.Y. Li & X.E. Xiao, conidiomata induced on PNA (ZJUE H-0028, culture CGMCC 3.20383 = BE 28); Zhejiang Province, Taizhou City, from the branch of C. reticulata cv. Succosa, 22 Jan. 2019, X.E. Xiao & Q.B. Huang, conidiomata induced on PNA (ZJUE H-0034, culture CGMCC 3.20384 = BE34); Zhejiang Province, Quzhou City, from the trunk of C. reticulata vs Ponkan, 23 Mar. 2018, H.K. Wang & X.E. Xiao, conidiomata induced on PNA (ZJUE H-0040, culture CGMCC3.20385 = BE40).

Notes — Phylogenetically, L. linhaiensis is closely related to L. acaciae, but can be separated from that species based on the length of its paraphyses. Paraphyses of L. linhaiensis (up to 80 μm long) are longer than those of L. acaciae (up to 69 μm long) (Zhang et al. 2021) (Table 4). Moreover, L. linhaiensis differs from L. acaciae by nucleotide differences in ITS (1 bp), tef1 (5 bp) and rpb2 loci (4 bp).

Lasiodiplodia ponkanicola X.E. Xiao, P.W. Crous & H.Y. Li, sp. nov. — MycoBank MB 840685; Fig. 13

Fig. 13.

Fig. 13

Lasiodiplodia ponkanicola. a. Pale yellow conidial mass oozing from conidioma formed on PNA; b. section view of conidioma; c–e. conidia developing on conidiogenous; f. hyaline, aseptate conidia; g–h. dark-brown, 1-septate conidia at two different focal planes to show the longitudinal striations; i. colony growing on PDA after 7 d. — Scale bars: a = 200 μm; b = 50 μm; c–h = 10 μm; i = 2.3 cm.

Etymology. Referring to the host variety (Ponkan) from which the fungus was isolated.

Typus. CHINA, Zhejiang Province, Quzhou City, from the trunk of C. reticulata cv. Ponkan, 23 Mar. 2018, H.K. Wang & X.E. Xiao, conidiomata induced on PNA (holotype ZJUE H-0044, culture ex-type CGMCC 3.20388 = BE44).

Sexual morph unknown. Conidiomata stromatic, produced on PNA within 2–4 wk, superficial or semi-immersed, dark brown to black, up to 1 mm diam, solitary or aggregated, unilocular, covered by mycelium, globose, thick-walled, often releasing pale yellow to saffron yellow conidial tendrils or mass. Paraphyses hyaline, cylindrical, septate, not branched, ends rounded, up to 87 μm long, 2–5 μm wide, formed among conidiogenous cells. Conidiophores absent. Conidiogenous cells holoblastic, hyaline, smooth, thin-walled, cylindrical, 8.5–40 × 2.5–9 μm. Conidia initially hyaline, aseptate, ellipsoid to ovoid, thin-walled with granular content, rounded at apex, base round or truncate, becoming pigmented, 1-septate with longitudinal striations, (16–)23.5–27.5(–28.5) × (11)–13–14.5(–15.5) μm (av. = 25.4 × 13.7 μm, n = 100; L/W ratio = 1.9) (Table 4).

Culture characteristics — Colonies on PDA with moderately dense aerial mycelium, initially white to smoke grey, turning grey olivaceous on the surface and greenish grey in reverse, becoming dark slate-blue with age. Colonies cover the 90 mm plates after 2 d in the dark at the optimum temperature of 25–30 °C. No growth was observed at 5 °C. After 2 d, colonies at 10 °C, 15 °C, 20 °C, 35 °C and 40 °C reach 9 mm, 19 mm, 43 mm, 71 mm and 26 mm, respectively.

Notes — Phylogenetically, L. ponkanicola is closely related to L. aquilariae (based on tef1), L. citricola (based on ITS and tub2), L. cinnamomic and L. huangyanensis (based on ITS/tef1/tub2/rpb2), but morphologically they can be separated on their average conidial dimensions and length of their paraphyses. Conidia of L. ponkanicola (av. 25.4 × 13.7; L/W = 1.9) are longer than L. cinnamomi (av. 19.9 × 13.4; L/W = 1.5) (Jiang et al. 2018) and L. citricola (av. 24.5 × 15.4; L/W = 1.6) (Abdollahzadeh et al. 2010), but shorter and narrower than L. aquilariae (av. 26.9 × 14.1; L/W = 1.8) (Wang et al. 2019) and L. huangyanensis (av. 30.1 × 15; L/W = 2) (this study). Moreover, the paraphyses of L. ponkanicola (up to 87 μm long) are longer than L. huangyanensis (up to 82 μm long) (this study) but shorter than L. aquilariae (up to 100 μm long) (Wang et al. 2019), L. cinnamomi (up to 106 μm long) (Jiang et al. 2018) and L. citricola (up to 125 μm long) (Abdollahzadeh et al. 2010) (Table 4). Furthermore, L. ponkanicola differs from these species by nucleotide differences in ITS (L. aquilariae: 3 bp, L. cinnamomi: 3 bp, L. huangyanensis: 3 bp), tef1 (L. aquilariae: 2 bp, L. cinnamomi: 6 bp, L. citricola: 7 bp, L. huangyanensis: 10 bp), tub2 (L. cinnamomi: 3 bp, L. huangyanensis: 3 bp) and rpb2 loci (L. aquilariae: 15 bp, L. cinnamomi: 9 bp, L. citricola: 15 bp). In view of the fact that isolate BE44 clustered with different species at different loci, it was considered that it may be a hybrid, which requires further research.

Sphaeropsis linhaiensis X.E. Xiao, P.W. Crous & H.Y. Li, sp. nov. — MycoBank MB 840686; Fig. 14

Fig. 14.

Fig. 14

Sphaeropsis linhaiensis. a. Conidiomata formed on PNA; b. section view of conidioma; c–d. conidia developing on conidiogenous cells; e–g. conidia; h. colony growing on PDA after 7 d. — Scale bars: a = 200 μm; b = 40 μm; c–g = 10 μm; h = 1.9 cm.

Etymology. Named after the Linhai City where it was isolated for the first time.

Typus. CHINA, Zhejiang Province, Linhai City, from a twig of C. unshiu, 2 June 2018, H.Y. Li, conidiomata induced on PNA (holotype ZJUE H-0018, culture ex-type CGMCC 3.20382 = BE18).

Sexual morph unknown. Conidiomata pycnidial, produced on PNA within 2–4 wk, dark brown to black, unilocular, up to 880 μm diam, immersed in the needle tissue, globose to subglobose, ostiolate, wall composed of several layers of dark brown textura angularis. Paraphyses hyaline, aseptate, up to 27 μm long, 1–5 μm wide. Conidiogenous cells hyaline, discrete, proliferating internally to form periclinal thickenings, 4.5–11 × 3–8 μm. Poor sporulation, conidia hyaline, aseptate, guttulate, oval to broadly ellipsoid, apex obtuse, base obtuse or truncate, moderately thick-walled, (26.5–)28.5–35(–38) × (11.5–)14–18(–19.5) μm (av. = 31.6 × 15.9 μm, n = 30; L/W ratio = 2) (Table 4).

Culture characteristics — Colonies on PDA initially white, turning olivaceous grey gradually on the surface and leaden grey at the reverse. Colonies cover the 90 mm plates after 6 d at the optimum temperature of 25 °C. No growth was observed at 5 °C, 35 °C and 40 °C. After 6 d, colonies at 10 °C, 15 °C, 20 °C and 30 °C reach 31 mm, 64 mm, 80 mm and 19 mm, respectively.

Notes — Phylogenetically, S. linhaiensis is closely related to S. citrigena, but can be distinguished from S. citrigena based on its average conidial dimensions. Conidia of S. linhaiensis (av. 31.6 × 15.9; L/W = 2) are longer than those of S. citrigena (av. 30.5 × 16.8; L/W = 1.8) (Phillips et al. 2008). Moreover, S. linhaiensis differs from S. citrigena by nucleotide differences in ITS (6 bp), tef1 (10 bp) and tub2 (7 bp).

Prevalence of Botryosphaeriaceae species

In total, 18 species of Botryosphaeriaceae were identified from citrus branch diseases in the Chongqing, Fujian, Guangdong, Guangxi, Hunan, Jiangxi, Shaanxi, Shanghai and Zhejiang provinces of China (Fig. 15a). These species include Botryosphaeria dothidea (32 isolates, 28.8 %), B. fabicerciana (4 isolates, 3.6 %), Diplodia seriata (1 isolate, 0.9 %), Dothiorella alpina (1 isolate, 0.9 %), Do. citrimurcotticola (6 isolates, 5.4 %), Do. plurivora (2 isolates, 1.8 %), Lasiodiplodia citricola (8 isolates, 7.2 %), L. guilinensis (2 isolates, 1.8 %), L. huangyanensis (3 isolates, 2.7 %), L. iraniensis (6 isolates, 5.4 %), L. linhaiensis (7 isolates, 6.3 %), L. microconidia (5 isolates, 4.5 %), L. ponkanicola (1 isolate, 0.9 %), L. pseudotheobromae (26 isolates, 23.4 %), L. theobromae (4 isolates, 3.6 %), Neodeightonia subglobosa (1 isolate, 0.9 %), Neofusicoccum parvum (1 isolate, 0.9 %) and Sphaeropsis linhaiensis (1 isolate, 0.9 %) (Fig. 15b). Of these 18 species, B. dothidea (28.8 %) was most commonly isolated, followed by L. pseudotheobromae (23.4 %), L. citricola (8 isolates, 7.2 %) and L. linhaiensis (7 isolates, 6.3 %) (Fig. 15a). In terms of the source of isolates, Zhejiang Province has the most isolates and the most diversity in species, most likely because of the more intensive sampling in this province (Fig. S8). In addition, L. pseudotheobromae was the most widely distributed species found in six provinces, including Chongqing, Fujian, Guangdong, Guangxi, Shaanxi and Zhejiang (Fig. 15b, S8).

Fig. 15.

Fig. 15

The prevalence of Botryosphaeriaceae species isolated from citrus. a. Distribution of Botryosphaeriaceae species in China; b. overall isolation rate (%) of Botryosphaeriaceae species. Different species are represented by numbers with different colours.

Pathogenicity tests

For in vitro inoculation, all 31 isolates inoculated were pathogenic to C. reticulata shoots with visible lesions. No lesions were produced on the shoots inoculated with PDA plugs (Fig. S9). Specifically, isolates of Lasiodiplodia spp. and Ne. parvum caused symptoms on all inoculated shoots (with incidence of 100 %), the isolates of D. seriata and Do. alpina caused symptoms on less than 60 % of the shoots, and the remaining isolates caused symptoms on shoots ranging from 60 to 100 % (Fig. 16a). Most isolates of Lasiodiplodia produced relatively longer lesions (mean lesion length > 15 cm) than the isolates from the other six genera. In contrast, isolates in N. subglobosa (5.09 cm), S. linhaiensis (5.64 cm), Do. alpina (7.06 cm) and D. seriata (7.75 cm) produced the shortest mean lesion lengths (Fig. 16b). In consideration of both disease incidence and lesion length, we concluded that species of Lasiodiplodia were more virulent to citrus than the other genera encountered.

Fig. 16.

Fig. 16

Pathogenicity tests results of inoculated isolates in Botryosphaeriaceae. a. Incidences of shoots inoculated in vitro and in vivo, the blue diamond represents the incidence of shoots inoculated in vitro, while the orange diamond represents the incidence of shoots inoculated in vivo; b. mean lesion lengths on C. reticulata shoots inoculated in vitro after 8 d. Bars represent standard errors. Columns with different letters indicate significant differences according to LSD test with confidence level α = 0.05. Control: PDA plugs.

For in vivo inoculation, all shoots of Cocktail grapefruit inoculated with the isolates listed in Fig. 16 produced lesions after 15 d inoculation, except for the isolates BE3 (B. fabicerciana) and BE17 (Do. alpina). Consistent with the results of in vitro inoculation, 100 % of the shoots inoculated with isolates of Lasiodiplodia spp. produced symptoms. The symptoms were similar to those observed in the field (Fig. S10a–g). However, the remaining isolates caused symptoms on less than 60 % of the shoots, except for isolates BE15 (100 %) and BE85 (80 %) (Fig. 16a). On the contrary, no necrosis symptoms were observed on the control shoots inspected 15 days after inoculation (Fig. 17h). All isolates inoculated in vitro and in vivo were re-isolated successfully from these lesions. As expected, no isolates of Botryosphaeriaceae were isolated from the control inoculations.

DISCUSSION

This study represents the first comprehensive characterisation of species in Botryosphaeriaceae isolated from citrus trees with twig and branch dieback, cankers and gummosis in China. Based on phylogenetic analyses and morphological characteristics, 18 species belonging to seven genera of Botryosphaeriaceae were identified. These species include Botryosphaeria dothidea, B. fabicerciana, Diplodia seriata, Dothiorella alpina, Do. plurivora, Lasiodiplodia citricola, L. iraniensis, L. microconidia, L. pseudotheobromae, L. theobromae, Neodeightonia subglobosa, Neofusicoccum parvum, and the new species described here, namely Do. citrimurcotticola, L. guilinensis, L. huangyanensis, L. linhaiensis, L. ponkanicola and Sphaeropsis linhaiensis. Of the 12 known species reported here, B. fabicerciana, Do. alpina, L. microconidia and N. subglobosa are reported on citrus in China for the first time.

Results of the pathogenicity tests indicate that all Lasiodiplodia species obtained in this study are pathogenic to the tested C. reticulata shoots in vitro and Cocktail grapefruit in vivo, with disease incidences of 100 %. Most of the remaining taxa were not as aggressive however, especially in the in vivo inoculation, which may be due to the higher ambient temperatures observed in the field (Zhang et al. 2016, 2021, this study). Moreover, significant differences in aggressiveness were observed among species. Most species of Lasiodiplodia were strongly aggressive. Conversely, N. subglobosa, S. linhaiensis, Do. alpina and D. seriata were weakly aggressive. In general, Lasiodiplodia was the most aggressive genus in the present study.

Botryosphaeria dothidea is the type species of Botryosphaeria and was considered as one of the most common and important pathogens of woody plants (Phillips et al. 2013, Marsberg et al. 2017). In this study, B. dothidea was the most commonly isolated species. Results of pathogenicity tests indicate that isolates of B. dothidea are moderately aggressive on C. reticulata shoots, which is similar to observations on Pistacia vera in California (Chen et al. 2014). Botryosphaeria fabicerciana, the other species of Botryosphaeria isolated in this study, was first reported on Eucalyptus and was weakly aggressive to this host (Chen et al. 2011). This is the first report of B. fabicerciana on citrus, and on C. reticulata it appeared to be mild to highly aggressive, while on Cocktail grapefruit it appeared to be weakly aggressive.

Diplodia seriata is regarded as a pathogen of citrus, causing branch canker and dieback in Algeria and the USA (Adesemoye et al. 2014, Berraf-Tebbal et al. 2020). Similarly, we isolated a single strain of D. seriata from C. sinensis with branch canker. This finding contrasts with that of Linaldeddu et al. (2015), who reported D. seriata to be the dominant species on Vitis in Italy. Pathogenicity tests indicate that D. seriata is less aggressive than most species of Botryosphaeriaceae obtained in this study.

Dothiorella gummosis refers to the occurrence of branch or trunk cankers on citrus caused by species of Botryosphaeriaceae. The pathogen was long believed to be Do. gregaria, the asexual morph of Botryosphaeria ribis (Adesemoye et al. 2011). However, other species that resided in the Botryosphaeriaceae were also found causing Dothiorella gummosis in citrus (Adesemoye et al. 2011). Therefore, the term ‘Dothiorella gummosis’ was no longer suitable for describing such symptoms, while ‘Botryosphaeria gummosis’ (Adesemoye et al. 2011) and ‘Bot gummosis’ (Adesemoye et al. 2014) were proposed as alternative. In this study, one undescribed Dothiorella species (Do. citrimurcotticola) and two previously reported species (Do. alpina and Do. plurivora) were isolated from branch dieback of Citrus spp. Dothiorella alpina was described from a dead tree of Platycladus orientalis in China (Zhang et al. 2016) and has not been reported from other hosts. Thus, this study represents the first report of this fungus on citrus. Dothiorella plurivora was first reported by Abdollahzadeh et al. (2014) in Iran and Spain, named for its broad host range, including twigs of Casuarina sp., Citrus sp., Cupressus sempervirens, Eucalyptus sp., Juglans regia, Malus domestica, Prunus armeniaca and Vitis vinifera. In this study, the isolates of Do. plurivora were collected from C. reticulata and C. unshiu with twig dieback. To our knowledge, this is the first report of Do. plurivora occurring on citrus in China. Pathogenicity tests demonstrated that the three species of Dothiorella were pathogenic to C. reticulata shoots, and that Do. alpina was less aggressive than the other two species. However, isolates of Do. citrimurcotticola and Do. plurivora were weakly aggressive on Cocktail grapefruit shoots, with incidences lower than 50 %, while Do. alpina was non-pathogenic on Cocktail grapefruit shoots.

We obtained nine species of Lasiodiplodia from citrus diseased branches, accounting for 55.9 % of the total number of isolates, making Lasiodiplodia the most prevalent genus with the highest number of species encountered in this study. This finding is consistent with previous reports that Lasiodiplodia is common on citrus (Abdollahzadeh et al. 2010, Adesemoye et al. 2014, Coutinho et al. 2017, Guajardo et al. 2018, Bautista-Cruz et al. 2019, Berraf-Tebbal et al. 2020). Probable reasons why species of Lasiodiplodia are dominant on citrus include the following: Firstly, it was observed that species of Lasiodiplodia are fast growing, covering 90 mm plates in only 2 d, while species of other genera of Botryosphaeriaceae have slower growth rates. Species of Lasiodiplodia also proved to be more aggressive to citrus compared to other genera tested. Secondly, the optimum temperature of the other genera in this study is 25 °C, while the optimum growth temperature of Lasiodiplodia spp. ranges from 25 °C to 30 °C, thereby giving it an advantage over other species at higher temperatures, and this is probably the reason why Lasiodiplodia species are mostly found in tropical or sub-tropical regions, and rare in regions with temperate climates.

Lasiodiplodia pseudotheobromae can infect citrus, causing gummosis, trunk canker, and twig blight (Abdollahzadeh et al. 2010, Bautista-Cruz et al. 2019, Ahmed et al. 2020). In the present study, L. pseudotheobromae was the second most abundant species isolated from citrus with symptoms of gummosis, dieback and canker. Furthermore, the results of pathogenicity tests showed that L. pseudotheobromae is one of the most aggressive species on citrus shoots. Stem-end rot is a common and economically important postharvest disease of citrus fruits worldwide, and it is usually thought to be caused by L. theobromae (Zhang 2014). Sultana et al. (2018) found that L. pseudotheobromae was also associated with citrus stem-end rot in Bangladesh. Stem-end rot is also an important postharvest disease on citrus in China (Cai et al. 2011). Since L. pseudotheobromae is similar to L. theobromae and is common on citrus according to the isolation results obtained in this study, it is possible that several reports of L. theobromae could have in fact been L. pseudotheobromae. In summary, L. pseudotheobromae is widely distributed on citrus, highly aggressive and can cause stem-end rot and branch diseases. Therefore, we consider L. pseudotheobromae to be an important pathogen on citrus in China, urgently requiring further research to elucidate its impact on this crop.

Neodeightonia subglobosa was initially reported on dead culms of Bambusa arundinacea in Sierra Leone (Punithalingam 1969). Furthermore, N. subglobosa was also found to cause keratomycosis in human eyes (Phillips et al. 2008). There are few reports about N. subglobosa, and to our knowledge, this study is the first to report N. subglobosa associated with gummosis on citrus. However, pathogenicity tests indicated that N. subglobosa was only weakly aggressive on citrus.

Neofusicoccum parvum has a broad host range and distribution, and has been reported from 90 hosts across six continents and 29 countries (Sakalidis et al. 2013, Batista et al. 2021). The lack of host specificity, combined with both a sexual and an asexual cycle, and the ability to live as a latent pathogen are conducive to the infection and spread of Ne. parvum (Sakalidis et al. 2013). In China, Ne. parvum was found associated with canker and dieback on Cupressus funebris (Li et al. 2010), Eucalyptus (Chen et al. 2011), Juglans regia (Yu et al. 2015), Prunus (Li et al. 2019, Zhang et al. 2019), Rhododendron (Yang et al. 2015) and Vitis heyneana (Wu et al. 2015). In this study, we found that Ne. parvum also caused dieback and gummosis on C. unshiu in China. Based on the pathogenicity tests, Ne. parvum was less aggressive than most of the species in this study, but disease incidences in vitro and in vivo were high, second only to isolates in Lasiodiplodia.

Previous research revealed Sphaeropsis citrigena to occur on dead bark of citrus (Phillips et al. 2013). In the present study, another species of Sphaeropsis, namely S. linhaiensis, was found that was associated with twig dieback on C. unshiu. Pathogenicity tests, however, indicated that S. linhaiensis is weakly aggressiveness on citrus.

Branch diseases of citrus are a persistent and frequent problem in the main citrus production areas of China. Many fungal pathogens can induce branch diseases on citrus, including species within the Botryosphaeriaceae, Diatrypaceae and genera such as Colletotrichum and Diaporthe (Chinese Academy of Agricultural Sciences 1960, Tai 1979, Cai et al. 2011). Because branch diseases are complex, and symptoms may be induced by multiple pathogens at the same time, there are few effective measures to control such diseases. Previous research has shown that wounds caused by pruning, mechanical injury, frost and sunburn damage have become the entry point for Botryosphaeriaceae on woody hosts (Savocchia et al. 2007, Úrbez-Torres & Gubler 2009, Eskalen et al. 2013). Furthermore, fungal pathogens release the greatest number of spores during and after rainfall events (Eskalen & Gubler 2001, Amponsah et al. 2009, Eskalen et al. 2013). Therefore, to prevent and reduce the occurrence of citrus branch diseases, pruning should avoid rainy days, decaying or dead branches and twigs should be removed from orchards, wounds protected with sealant or fungicides, frost damage avoided where possible and trees protected from the sun in hot weather. Furthermore, because species of Botryosphaeriaceae are latent pathogens, they can become pathogenic when the trees are under stress or in weak vigour (Slippers & Wingfield 2007). Hence, the cultivation of good tree vigour is conducive to enhance disease resistance.

In conclusion, results of this study present the first detailed research of 18 species of Botryosphaeriaceae causing branch diseases on citrus in nine major citrus-producing provinces of China. Overall, Lasiodiplodia was found to be the most prevalent and aggressive genus in this study, which indicates that Lasiodiplodia is one of the most important genera causing citrus branch diseases. Besides, L. pseudotheobromae is one of the most abundant and most prevalent species, which together with its aggressiveness in branches and fruits, makes L. pseudotheobromae an economically important pathogen on citrus. To better prevent and control L. pseudotheobromae, further research is needed to study the relationship between L. pseudotheobromae in different regions and different citrus varieties, and its ability to cause stem-end rot. In the current study, several species were obtained as single isolates, but most of the isolates were from Zhejiang Province, so subsequent sampling needs to be expanded to investigate the prevalence of these species in other citrus-producing areas in China. Because species of Botryosphaeriaceae can be latent pathogens in woody host plants, and jump from hosts planted nearby (Damm et al. 2007, Slippers & Wingfield 2007, Begoude et al. 2012, James et al. 2017), collecting plant hosts adjacent to citrus is also useful for studying the diversity of the Botryosphaeriaceae species that could have an impact on citrus cultivation.

Acknowledgments

This research was supported by the Key Research and Development Program of Zhejiang Province (2019C02022), the National Key Research and Development Program (2017YFD0202000) and the China Agriculture Research System (CARS-26). We thank the China Eucalypt Research Centre (CERC) for technical guidance in the process of species identification, including phylogenetic analysis, morphology description and pathogenicity tests. We thank Mr. Qu Wu, Siqing Zhao, Dekuan Ding, Qianbin Huang, Weilong Li and Ms. Lan Cheng for their assistance during sample collection. We thank Mr. Xielong Yu for providing the citrus shoots for pathogenicity tests.

Supplementary material

Fig. S1

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Botryosphaeria. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Neofusicoccum parvum (CMW 9081).

per-2023-47-3-SF1-1.jpg (547.7KB, jpg)
per-2023-47-3-SF1-2.jpg (483.2KB, jpg)
Fig. S2

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Diplodia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Lasiodiplodia theobromae (CBS 164. 96).

per-2023-47-3-SF2-1.jpg (688.4KB, jpg)
per-2023-47-3-SF2-2.jpg (714.7KB, jpg)
per-2023-47-3-SF2-3.jpg (593.5KB, jpg)
Fig. S3

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Dothiorella. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Neofusicoccum parva (CMW 9081).

per-2023-47-3-SF3-1.jpg (770.7KB, jpg)
per-2023-47-3-SF3-2.jpg (820.7KB, jpg)
per-2023-47-3-SF3-3.jpg (684.1KB, jpg)
Fig. S4

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Lasiodiplodia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and type species are in red bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

per-2023-47-3-SF4-1.jpg (548.3KB, jpg)
per-2023-47-3-SF4-2.jpg (579.1KB, jpg)
per-2023-47-3-SF4-3.jpg (501.6KB, jpg)
per-2023-47-3-SF4-4.jpg (537.6KB, jpg)
Fig. S5

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1 and tub2 (a–c) sequence alignments of Neodeightonia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CBS 115476).

per-2023-47-3-SF5.jpg (538.1KB, jpg)
Fig. S6

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Neofusicoccum. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Dothiorella viticola (CBS 117009).

per-2023-47-3-SF6-1.jpg (874.3KB, jpg)
per-2023-47-3-SF6-2.jpg (789.4KB, jpg)
per-2023-47-3-SF6-3.jpg (836.8KB, jpg)
per-2023-47-3-SF6-4.jpg (616.5KB, jpg)
Fig. S7

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Sphaeropsis. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

per-2023-47-3-SF7-1.jpg (424.5KB, jpg)
Fig. S8

The distribution of Botryosphaeriaceae species obtained from nine provinces. Provinces are represented by different colours.

per-2023-47-3-SF8.jpg (211.1KB, jpg)
Fig. S9

Symptoms developed in the detached shoots of C. reticulata inoculated with isolates in Botryosphaeriaceae 8 d after inoculation. B. dothidea: BE1, BE2; B. fabicerciana: BE3, BE85; D. seriata: BE4; Do. alpina: BE17; Do. citrimurcotticola: BE5, BE8; Do. plurivora: BE16, BE74; L. citricola: BE13, BE38; L. guilinensis: BE31, BE59; L. huangyanensis: BE33, BE50; L. iranensis: BE27, BE41, BE100; L. linhaiensis: BE51, BE28; L. microconidia: BE80, BE88; L. ponkanicola: BE44; L. pseudotheobromae: BE10, BE11; L. theobromae: BE20, BE21; N. subglobosa: BE14; Ne. parvum: BE15; S. linhaiensis: BE18.

per-2023-47-3-SF9.jpg (805KB, jpg)
Fig. S10

Symptoms developed in shoots of Cocktail grapefruit plants inoculated with isolates of Botryosphaeriaceae 15 d after inoculation. a–b. Shoots inoculated with B. fabicerciana (BE85) producing gum exudate; c. gummosis caused by Ne. parvum (BE15); d. shoot showing symptoms of dieback and gummosis after inoculation with L. guilinensis (BE59); e. dieback with a large amount of gummosis caused by L. pseudotheobromae (BE10); f. dieback with gummosis caused by L. huangyanensis (BE50); g. conidiomata formed on the inoculated shoots with dieback; h. shoots inoculated with sterile PDA plugs. Red arrows indicate the inoculated positions; orange arrows indicate gum exudate; white arrow indicates a conidioma.

per-2023-47-3-SF10.jpg (1.5MB, jpg)

REFERENCES

  1. Abdollahzadeh J, Javadi A, Mohammadi Goltapeh E, et al. 2010. Phylogeny and morphology of four new species of Lasiodiplodia from Iran. Persoonia 25: 1–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abdollahzadeh J, Javadi A, Zare R, et al. 2014. Phylogenetic study of Dothiorella and Spencermartinsia species associated with woody plants in Iran, New Zealand, Portugal and Spain. Persoonia 32: 1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Adesemoye AO, Eskalen A. 2011. First report of Spencermartinsia viticola, Neofusicoccum australe, and N. parvum causing branch canker of citrus in California. Plant Disease 95: 770. [DOI] [PubMed] [Google Scholar]
  4. Adesemoye AO, Eskalen A, Faber B, et al. 2011. Multiple Botryosphaeria species causing ‘Dothiorella’ gummosis in citrus. Citrograph 32–34. [Google Scholar]
  5. Adesemoye AO, Mayorquin JS, Wang DH, et al. 2014. Identification of species of Botryosphaeriaceae causing bot gummosis in citrus in California. Plant Disease 98: 55–61. [DOI] [PubMed] [Google Scholar]
  6. Ahmed MZ, Shafique MS, Anwaar HMA, et al. 2020. First report of Lasiodiplodia pseudotheobromae causing trunk cankers in Citrus reticulata blanco (Kinnow) in Pakistan. Plant Disease 104: 2522. [Google Scholar]
  7. Alves A, Correia A, Luque J, et al. 2004. Botryosphaeria corticola, sp. nov. on Quercus species, with notes and description of Botryosphaeria stevensii and its anamorph, Diplodia mutila. Mycologia 96: 598–613. [PubMed] [Google Scholar]
  8. Alves A, Crous PW, Correia A, et al. 2008. Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Diversity 28: 1–13. [Google Scholar]
  9. Amponsah NT, Jones EE, Ridgway HJ, et al. 2009. Rainwater dispersal of Botryosphaeria conidia from infected grapevines. New Zealand Plant Protection 62: 228–233. [Google Scholar]
  10. Azadeh G, Abdoolnabi B, Majeed AS, et al. 2018. Citrus × aurantiifolia, a new host report of Macrophomina phaseolina in Iran. Australasian Plant Disease Notes 13: 15. [Google Scholar]
  11. Batista E, Lopes A, Alves A. 2021. What do we know about Botryosphaeriaceae? An overview of a worldwide cured dataset. Forests 12: 313–330. [Google Scholar]
  12. Bautista-Cruz MA, Almaguer-Vargas G, Leyva-Mir SG, et al. 2019. Phylogeny, distribution, and pathogenicity of Lasiodiplodia species associated with cankers and dieback symptoms of Persian lime in Mexico. Plant Disease 103: 1156–1165. [DOI] [PubMed] [Google Scholar]
  13. Begoude BAD, Slippers B, Perez G, et al. 2012. High gene flow and outcrossing within populations of two cryptic fungal pathogens on a native and non-native host in Cameroon. Fungal Biology 116: 343–353. [DOI] [PubMed] [Google Scholar]
  14. Berraf-Tebbal A, Mahamedi AE, Aigoun-Mouhous W, et al. 2020. Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria. PLOS ONE 15: e0232448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Brown GE, Eckert JW. 2000. Diplodia stem-end rot. In: Timmer LW, Gernsey SM, Graham JH. (eds), Compendium of citrus diseases, second ed: 43–44. APS Press, St Paul. [Google Scholar]
  16. Cai MD, Yi GJ, Peng CJ. 2011. An illustrated book of primary colours of citrus diseases and pests. China Agricultural Press. [In Chinese.] [Google Scholar]
  17. Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553–556. [Google Scholar]
  18. Chen SF, Liu QL, Li GQ, et al. 2018. A new genus of Cryphonectriaceae isolated from Lagerstroemia speciosa in southern China. Plant Pathology 67: 107–123. [Google Scholar]
  19. Chen SF, Morgan DP, Michailides TJ. 2014. Botryosphaeriaceae and Diaporthaceae associated with panicle and shoot blight of pistachio in California, USA. Fungal Diversity 67: 157–179. [Google Scholar]
  20. Chen SF, Pavlic D, Roux J, et al. 2011. Characterization of Botryosphaeriaceae from plantation-grown Eucalyptus species in South China. Plant Pathology 60: 739–751. [Google Scholar]
  21. Cheng J, Wei X, Fan H. 2004. Phytophthora species infecting citrus in Guangdong Province. Journal of South China Agricultural University (Natural Science Edition) 25: 31–33. [In Chinese.] [Google Scholar]
  22. Chinese Academy of Agricultural Sciences. 1960. Chinese journal of fruit diseases and insects. Agricultural Press. [In Chinese.] [Google Scholar]
  23. Coutinho IBL, Freire FCO, Lima CS, et al. 2017. Diversity of genus Lasiodiplodia associated with perennial tropical fruit plants in Northeastern Brazil. Plant Pathology 66: 90–104. [Google Scholar]
  24. Cruywagen EM, Slippers B, Roux J, et al. 2017. Phylogenetic species recognition and hybridisation in Lasiodiplodia: a case study on species from baobabs. Fungal Biology 121: 420–436. [DOI] [PubMed] [Google Scholar]
  25. Damm U, Crous PW, Fourie PH. 2007. Botryosphaeriaceae as potential pathogens of Prunus in South Africa, with descriptions of Diplodia africana and Lasiodiplodia plurivora sp. nov. Mycologia 99: 664–680. [DOI] [PubMed] [Google Scholar]
  26. Darriba D, Taboada GL, Doallo R, et al. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Deng XX, Peng CJ, Chen ZS, et al. 2008. Chinese citrus varieties. China Agricultural Press. [In Chinese.] [Google Scholar]
  28. Eskalen A, Faber B, Bianchi M. 2013. Spore trapping and pathogenicity of fungi in the Botryosphaeriaceae and Diaporthaceae associated with avocado branch canker in California. Plant Disease 97: 329–332. [DOI] [PubMed] [Google Scholar]
  29. Eskalen A, Gubler W. 2001. Association of spores of Phaeomoniella chlamydospora, Phaeoacremonium inflatipes, and Pm. aleophilum with grapevine cordons in California. Phytopathologia Mediterranea 40: 429–432. [Google Scholar]
  30. FAO. 2018. Food and Agriculture Organization of the United Nations, Statistical Databases. http://www.fao.org. [Google Scholar]
  31. Fawcett HS, Burger OF. 1911. A gum-inducing Diplodia of peach and orange. Mycologia 3: 151–153. [Google Scholar]
  32. Glass NL, Donaldson GC. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61: 1323–1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Guajardo J, Riquelme N, Tapia L, et al. 2018. First Report of Lasiodiplodia theobromae causing bot gummosis in citrus limon in Chile. Plant Disease 102: 818. [Google Scholar]
  34. Guindon S, Dufayard JF, Lefort V, et al. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321. [DOI] [PubMed] [Google Scholar]
  35. Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic Biology 42: 182–192. [Google Scholar]
  36. Huang F, Chen GQ, Hou X, et al. 2013a. Colletotrichum species associated with cultivated citrus in China. Fungal Diversity 61: 61–74. [Google Scholar]
  37. Huang F, Hou X, Dewdney M, et al. 2013b. Diaporthe species occurring on citrus in China. Fungal Diversity 61: 237–250. [Google Scholar]
  38. Huang F, Zhu L, Hou X, et al. 2012. Identification of the pathogenic fungus causing brown spot on Ougan. Journal of Zhejiang Agricultural Sciences 1281–1282. [In Chinese.] [Google Scholar]
  39. Ismail MA, Zhang JX. 2004. Post-harvest citrus diseases and their control. Outlooks Pest Management 15: 29–35. [Google Scholar]
  40. James WMM, Bernard S, Jolanda R, et al. 2017. Overlap of latent pathogens in the Botryosphaeriaceae on a native and agricultural host. Fungal Biology 121: 405–419. [DOI] [PubMed] [Google Scholar]
  41. Jiang N, Wang XE, Liang YM, et al. 2018. Lasiodiplodia cinnamomi sp. nov. from Cinnamomum camphora in China. Mycotaxon 133: 249–259. [Google Scholar]
  42. Katoh K, Rozewicki J, Yamada KD. 2019. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20: 1160–1166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Li QL, Tang LH, Sun WX, et al. 2019. First report of stem canker and dieback caused by Neofusicoccum parvum on Plum in Guangxi, Southern China. Plant Disease 103: 2952–2952. [Google Scholar]
  45. Li SB, Li JZ, Lu ZH, et al. 2010. First report of Neofusicoccum parvum causing dieback disease of Chinese Weeping Cypress in China. Plant Disease 94: 641–641. [DOI] [PubMed] [Google Scholar]
  46. Linaldeddu BT, Deidda A, Scanu B, et al. 2015. Diversity of Botryosphaeriaceae species associated with grapevine and other woody hosts in Italy, Algeria and Tunisia, with descriptions of Lasiodiplodia exigua and Lasiodiplodia mediterranea sp. nov. Fungal Diversity 71: 201–214. [Google Scholar]
  47. Liu YJ, Whelen S, Hall BD. 1999. Phylogenetic relationships among Ascomycetes: evidence from an RNA polymerse II subunit. Molecular Biology and Evolution 16: 1799–1808. [DOI] [PubMed] [Google Scholar]
  48. Marsberg A, Kemler M, Jami F, et al. 2017. Botryosphaeria dothidea: a latent pathogen of global importance to woody plant health. Molecular Plant Pathology 18: 477–488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Mayorquin JS, Wang DH, Twizeyimana M, et al. 2016. Identification, distribution, and pathogenicity of Diatrypaceae and Botryosphaeriaceae associated with citrus branch canker in the Southern California desert. Plant Disease 100: 2402–2413. [DOI] [PubMed] [Google Scholar]
  50. Pérez CA, Wingfield MJ, Slippers B, et al. 2010. Endophytic and canker-associated Botryosphaeriaceae occurring on non-native Eucalyptus and native Myrtaceae trees in Uruguay. Fungal Diversity 41: 53–69. [Google Scholar]
  51. Phillips AJL, Alves A, Abdollahzadeh J, et al. 2013. The Botryosphaeriaceae: genera and species known from culture. Studies in Mycology 76: 51–167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Phillips AJL, Alves A, Pennycook SR, et al. 2008. Resolving the phylogenetic and taxonomic status of dark-spored teleomorph genera in the Botryosphaeriaceae. Persoonia 21: 29–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Polizzi G, Aiello A, Vitale A, et al. 2009. First report of shoot blight, canker, and gummosis caused by Neoscytalidium dimidiatum on citrus in Italy. Plant Disease 93: 1215. [DOI] [PubMed] [Google Scholar]
  54. Punithalingam E. 1969. Studies on Sphaeropsidales in culture. Mycological Papers 119: 1–24. [Google Scholar]
  55. Qin X, Deng M, Yang T, et al. 2012. Pathogen identification of black rot of Gong’an. South China Fruits 41: 10–13. [In Chinese.] [Google Scholar]
  56. Rayner RW. 1970. A Mycological Colour Chart. Kew, Commonwealth Mycological Institute. [Google Scholar]
  57. Sakalidis ML, Slippers B, Wingfield BD, et al. 2013. The challenge of understanding the origin, pathways and extent of fungal invasions: global populations of the Neofusicoccum parvum-N. ribis species complex. Diversity and Distributions 19: 873–883. [Google Scholar]
  58. Savocchia S, Steel CC, Stodart BJ, et al. 2007. Pathogenicity of Botryosphaeria species isolated from declining grapevines in subtropical regions of Eastern Australia. Vitis 46: 27–32. [Google Scholar]
  59. Schindelin J, Arganda-Carreras I, Frise E, et al. 2012. Fiji: an open-source platform for biological-image analysis. Nature Methods 9: 676–682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Schoch CL, Shoemaker RA, Seifert KA, et al. 2006. A multigene phylogeny of the Dothideomycetes using four nuclear loci. Mycologia 98: 1041–1052. [DOI] [PubMed] [Google Scholar]
  61. Shen ZM. 2019. Present status and prospects of citrus production in China. Scientific Breeding 9: 5–10. [In Chinese.] [Google Scholar]
  62. Slippers B, Crous PW, Jami F, et al. 2017. Diversity in the Botryosphaeriales: Looking back, looking forward. Fungal Biology 121: 307–321. [DOI] [PubMed] [Google Scholar]
  63. Slippers B, Wingfield MJ. 2007. Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity, ecology and impact. Fungal Biology Reviews 21: 90–106. [Google Scholar]
  64. Smith CO. 1934. Inoculations showing the wide host range of Botryosphaeria ribis. Journal of Agricultural Research 49: 467–476. [Google Scholar]
  65. Smith H, Wingfield MJ, Crous PW, et al. 1996. Sphaeropsis sapinea and Botryosphaeria dothidea endophytic in Pinus spp. and Eucalyptus spp. in South Africa. South African Journal of Botany 62: 86–88. [Google Scholar]
  66. SPSS. 2011. IBM SPSS Statistics Base 20. IBM Corporation, Armonk, NY. [Google Scholar]
  67. Sultana R, Islam MS, Rahman H, et al. 2018. Characterization of Lasiodiplodia pseudotheobromae associated with citrus stem-end rot disease in Bangladesh. International Journal of Biosciences 13: 252–262. [Google Scholar]
  68. Swofford DL. 2003. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Version 4.0b10. Sinauer Associates, Sunderland, MA, USA. [Google Scholar]
  69. Tai FL. 1979. Sylloge Fungorum Sinicorum. Beijing, Science Press. [In Chinese.] [Google Scholar]
  70. Theissen F, Sydow H. 1918. Vorentwürfe zu den Pseudosphaeriales. Annales Mycologici 16: 1–34. [Google Scholar]
  71. Úrbez-Torres JR. 2011. The status of Botryosphaeriaceae species infecting grapevines. Phytopathologia Mediterranea 50: S5–S45. [Google Scholar]
  72. Úrbez-Torres JR, Gubler WD. 2009. Pathogenicity of Botryosphaeriaceae species isolated from grapevine cankers in California. Plant Disease 93: 584–592. [DOI] [PubMed] [Google Scholar]
  73. Van Burik JAH, Schreckhise RW, White TC, et al. 1998. Comparison of six extraction techniques for isolation of DNA from filamentous fungi. Medical Mycology 36: 299–303. [PubMed] [Google Scholar]
  74. Wang Y, Lin S, Zhao L, et al. 2019. Lasiodiplodia spp. associated with Aquilaria crassna in Laos. Mycological Progress 18: 683–701. [Google Scholar]
  75. White TJ, Bruns T, Lee S, et al. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, et al. (eds), PCR protocols: a guide to methods and applications: 315–322. Academic Press, San Diego, CA, USA. [Google Scholar]
  76. Wu DD, Fu G, Ye YF, et al. 2015. First report of Neofusicoccum parvum causing panicle blight and leaf spot on Vitis heyneana in China. Plant Disease 99: 417–417. [DOI] [PubMed] [Google Scholar]
  77. Yang T, Groenewald JZ, Cheewangkoon R, et al. 2017. Families, genera, and species of Botryosphaeriales. Fungal Biology 2016: 322–346. [DOI] [PubMed] [Google Scholar]
  78. Yang XM, Wang JH, Zhang Y, et al. 2015. First report of Neofusicoccum parvum causing stem canker and dieback in Rhododendron in China. Plant Disease 99: 1179–1180. [Google Scholar]
  79. Yu ZD, Tang GH, Peng SB, et al. 2015. Neofusicoccum parvum causing canker of seedlings of Juglans regia in China. Journal of Forestry Research 26: 1019–1024. [Google Scholar]
  80. Zhang JX. 2014. Lasiodiplodia theobromae in citrus fruit (Diplodia stem-end rot). In: Bautista-Baños S, Postharvest decay: 309–335. Academic Press. [Google Scholar]
  81. Zhang LQ, Li XW, Su MS, et al. 2019. First report of Neofusicoccum parvum associated with shoot cankers of peach (Prunus persica) in Shanghai, China. Journal of Plant Pathology 101: 1257. [Google Scholar]
  82. Zhang M, He W, Wu JR, et al. 2016. Two new species of Spencermartinsia (Botryosphaeriaceae, Botryosphaeriales) from China. Mycosphere 7: 942–949. [Google Scholar]
  83. Zhang W, Groenewald JZ, Lombard L, et al. 2021. Evaluating species in Botryosphaeriales. Persoonia 46: 63–115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  84. Zhu L, Zhao XL, Wu RC, et al. 2011. Identification of the pathogen causing foot rot of Fortunella margarita (Lour.) Swingle. Acta Phytopathologica Sinica 41: 631–634. [In Chinese.] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Botryosphaeria. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Neofusicoccum parvum (CMW 9081).

per-2023-47-3-SF1-1.jpg (547.7KB, jpg)
per-2023-47-3-SF1-2.jpg (483.2KB, jpg)
Fig. S2

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Diplodia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Lasiodiplodia theobromae (CBS 164. 96).

per-2023-47-3-SF2-1.jpg (688.4KB, jpg)
per-2023-47-3-SF2-2.jpg (714.7KB, jpg)
per-2023-47-3-SF2-3.jpg (593.5KB, jpg)
Fig. S3

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Dothiorella. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Neofusicoccum parva (CMW 9081).

per-2023-47-3-SF3-1.jpg (770.7KB, jpg)
per-2023-47-3-SF3-2.jpg (820.7KB, jpg)
per-2023-47-3-SF3-3.jpg (684.1KB, jpg)
Fig. S4

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Lasiodiplodia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and type species are in red bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

per-2023-47-3-SF4-1.jpg (548.3KB, jpg)
per-2023-47-3-SF4-2.jpg (579.1KB, jpg)
per-2023-47-3-SF4-3.jpg (501.6KB, jpg)
per-2023-47-3-SF4-4.jpg (537.6KB, jpg)
Fig. S5

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1 and tub2 (a–c) sequence alignments of Neodeightonia. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CBS 115476).

per-2023-47-3-SF5.jpg (538.1KB, jpg)
Fig. S6

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Neofusicoccum. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Dothiorella viticola (CBS 117009).

per-2023-47-3-SF6-1.jpg (874.3KB, jpg)
per-2023-47-3-SF6-2.jpg (789.4KB, jpg)
per-2023-47-3-SF6-3.jpg (836.8KB, jpg)
per-2023-47-3-SF6-4.jpg (616.5KB, jpg)
Fig. S7

Phylogenetic trees generated by maximum likelihood analyses based on the individual ITS, tef1, tub2 and rpb2 (a–d) sequence alignments of Sphaeropsis. Bootstrap support values ≥ 50 % for ML and MP are presented above branches as follows: ML/MP bootstrap values < 50 % are marked with *, and absent are marked with -. Newly generated sequences are in red and ex-type strains are in bold. The tree was rooted to Botryosphaeria dothidea (CMW 8000).

per-2023-47-3-SF7-1.jpg (424.5KB, jpg)
Fig. S8

The distribution of Botryosphaeriaceae species obtained from nine provinces. Provinces are represented by different colours.

per-2023-47-3-SF8.jpg (211.1KB, jpg)
Fig. S9

Symptoms developed in the detached shoots of C. reticulata inoculated with isolates in Botryosphaeriaceae 8 d after inoculation. B. dothidea: BE1, BE2; B. fabicerciana: BE3, BE85; D. seriata: BE4; Do. alpina: BE17; Do. citrimurcotticola: BE5, BE8; Do. plurivora: BE16, BE74; L. citricola: BE13, BE38; L. guilinensis: BE31, BE59; L. huangyanensis: BE33, BE50; L. iranensis: BE27, BE41, BE100; L. linhaiensis: BE51, BE28; L. microconidia: BE80, BE88; L. ponkanicola: BE44; L. pseudotheobromae: BE10, BE11; L. theobromae: BE20, BE21; N. subglobosa: BE14; Ne. parvum: BE15; S. linhaiensis: BE18.

per-2023-47-3-SF9.jpg (805KB, jpg)
Fig. S10

Symptoms developed in shoots of Cocktail grapefruit plants inoculated with isolates of Botryosphaeriaceae 15 d after inoculation. a–b. Shoots inoculated with B. fabicerciana (BE85) producing gum exudate; c. gummosis caused by Ne. parvum (BE15); d. shoot showing symptoms of dieback and gummosis after inoculation with L. guilinensis (BE59); e. dieback with a large amount of gummosis caused by L. pseudotheobromae (BE10); f. dieback with gummosis caused by L. huangyanensis (BE50); g. conidiomata formed on the inoculated shoots with dieback; h. shoots inoculated with sterile PDA plugs. Red arrows indicate the inoculated positions; orange arrows indicate gum exudate; white arrow indicates a conidioma.

per-2023-47-3-SF10.jpg (1.5MB, jpg)

Articles from Persoonia : Molecular Phylogeny and Evolution of Fungi are provided here courtesy of Naturalis Biodiversity Center & Centraalbureau voor Schimmelcultures

RESOURCES