Skip to main content
NCBI home page
Journal of Fungi logoLink to Journal of Fungi
. 2025 Sep 22;11(9):686. doi: 10.3390/jof11090686

Botryosphaeriaceae Species Causing Stem Blight and Dieback of Blueberries in Serbia

Miloš Marić 1, Mira Vojvodić 2, Darko Jevremović 3, Bojana Vasilijević 3, Tanja Vasić 4, Miljan Grkinić 2, Aleksandra Bulajić 2,*
Editor: Nengguo Tao
PMCID: PMC12470446  PMID: 41003232

Abstract

In the main growing areas in Serbia, plants with symptoms of stem blight were sampled in nine orchards with American highbush blueberry (Vaccinium corymbosum), cultivar ‘Duke’, with high disease incidence, and 153 samples were taken. A total of 128 Botryosphaeriaceae isolates were characterized on the basis of morphology, sequence analysis, multilocus phylogeny based on ITS, TEF1-α and TUB2 sequences and pathogenicity, and belonged to one of the four species Neofusicoccum parvum, Botryosphaeria dothidea, Diplodia seriata and Lasiodiplodia iraniensis. Both D. seriata and L. iraniensis were detected for the first time on blueberries in Serbia, and L. iraniensis was detected for the first time on blueberries worldwide. Comparative morphological and TEF1-α sequence analyses allowed a clear separation of L. iraniensis from the phylogenetically closely related L. fujianensis, L. thailandica and L. endophytica. Of the nine blueberry cultivars ‘Aurora’, ‘Barbara Ann’, ‘Bluecrop’, ‘Bluejay’, ‘Draper’, ‘Duke’, ‘Huron’, ‘Patriot’ and ‘Spartan’ inoculated with L. iraniensis (isolate 421-19), the cultivar ‘Duke’ was the most susceptible. In our study, the majority of orchards were in their second or third year of production, implying that the planting material is likely to be the source of infection, emphasizing the importance of pathogen-free planting material.

Keywords: diagnosis, morphology, phylogeny, cultivar susceptibility

1. Introduction

The American highbush blueberry (Vaccinium corymbosum L., Fam. Ericaceae) is commercially cultivated worldwide under various climatic conditions [1] as its fruits have exceptional nutritional properties and positive effects on health [2]. As it is a very profitable crop, the global production of blueberries is constantly increasing, with more than 1.2 million tonnes being produced in 2022, and America being the largest producer (979,668 tonnes), followed by Europe (207,915 tonnes) (https://www.fao.org/faostat, accessed on 15 May 2025). In Serbia, the production of highbush blueberries is also increasing very rapidly, much faster than the production of any other fruit species [3] and is currently grown on over 2500 ha [4].

Blueberry production worldwide can be affected by a number of biotic and abiotic factors, and among these, fungi from the Botryosphaeriaceae family are considered one of the most important and devastating factors limiting blueberry production [5,6,7,8,9]. In New Zealand, diseases caused by Botryosphaeriaceae are considered particularly devastating in newly planted orchards, where nearly 20% of plants are infected and significant annual costs are incurred due to yield loss and replanting costs [6]. The most important factor contributing to the rapid spread of Botryosphaeriaceae disease is probably related to the health status of the planting material [10]. Due to the broad host range, latent infections, the ability to infect plants via wounds and the limited possibilities of efficient disease control [11], Botryosphaeriaceae pose a major challenge to the production of numerous host plants, including blueberries. The fungal family Botryosphaeriaceae currently comprises 24 defined genera with diverse lifestyles, saprobes, endophytes and pathogens associated with a wide range of host plants. Among the Botryosphaeriaceae, the genera Botryosphaeria, Diplodia, Lasiodiplodia, Neofusicoccum, Dothiorella and Neoscytalidium are the most important and best-studied plant pathogens [12].

Although previously considered a possible synonym of Diplodia [13], the fungal genus Lasiodiplodia Ellis & Everh has long been recognized and is well defined based on the morphology of the pycnidia, the longitudinal striation of the mature conidia and phylogenetic studies [14,15]. Lasiodiplodia is a very dynamic genus with over 47 established species to date, with new species being described relatively frequently [16,17]. Some Lasiodiplodia species are even considered to be of quarantine importance, such as L. pseudotheobromae [18] and more recently, L. iraniensis [19]. Lasiodiplodia iraniensis is a relatively newly described species that occurs as a pathogen of Salvadora persica, Juglans spp., mango, Eucalyptus spp., Citrus spp. and tropical almonds in Iran [20]. Several studies have shown that the status of isolates identified as L. jatrophicola as a closely related but distinct species from L. iraniensis is not justified, and it is currently synonymized with L. iraniensis [19,21,22,23]. After the initial description, L. iraniensis was found on mango in Western Australia [24], the United Arab Emirates [25], Brazil [26] and Peru [22], on mandarins in the United Arab Emirates [25], Bougainvillea spectabilis in southern China [27], Anacardium occidentale in Brazil [28], Persian lime in Mexico [23] and, more recently, on Adansonia digitata in Mozambique [29], Eucaliptus in India [30], bananas in Brazil [31] and yam and sweet oranges [19,32] in the USA. In all these regions and on all host plants, L. iraniensis has been described as an aggressive and economically important pathogen.

The intensive increase in blueberry production in Serbia has been accompanied by the appearance of various symptoms of stem blight of a largely unknown origin, which has triggered research that has recently confirmed the presence of blueberry strain pathogens including Macrophomina phaseolina [33], Fusarium sporotrichioides [34], Neopestalotiopsis clavispora [35] and N. vaccinii, N. rosae, Diaporthe eres, D. foeniculina and Neofusicoccum parvum [36]. During the study of blueberry stem diseases, we obtained a considerable number of Botryosphaeriaceae isolates from symptomatic plants, and our main objectives were as follows: (i) to identify the causal pathogens; (ii) to investigate morphological characteristics and the ability to grow at extreme temperatures; (iii) to determine the taxonomic position of the obtained isolates based on the sequences of ITS rDNA, translation elongation factor 1α (TEF1-α) and β-tubulin (TUB2); (iv) to determine the phylogenetic relationship between the Botryosphaeriaceae and, in particular, between the Lasiodiplodia spp. isolates and the relationship with newly detected species in Serbia; and (v) to evaluate the susceptibility of nine blueberry cultivars grown in Serbia and worldwide to selected discovered Lasiodiplodia sp.

2. Materials and Methods

2.1. Sampling and Isolations

The blueberry stem disease survey was conducted from 2011 to 2022, and the field inspections covered nine production fields/locations in four administrative districts in Serbia. The blueberry cultivar ‘Duke’ was grown in all orchards and a total of 153 samples were collected (Table 1). Disease incidence was calculated in each orchard (by zigzag inspections and random assessment of 100 plants in three replicates), and 5–25 samples were collected, depending on size of the orchard and symptoms. Small woody fragments of necrotic tissue were taken from each sample, surface sterilized with 2% sodium hypochlorite, placed on potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 17 g agar and 1 litre distilled H2O) [37] and incubated at 24 °C for 5 days. One or more representative colonies with the same morphology were selected from each of the nine growing fields, from which monosporial isolates were obtained for further characterization. The isolates were stored on sealed PDA slants at 4 °C in the fungal collection of the Department of Phytopathology, Institute of Phytomedicine, University of Belgrade—Faculty of Agriculture.

Table 1.

Geographic distribution of isolates collected in Serbia.

No. * Year District Locality Blueberry
Cultivar		 Estimated Disease Incidence (%) No. of Collected Samples Fungal Species Detected
(Positive Samples)		 Isolates
Culture ITS Seq
1 2011 Kolubara Belanovica Duke 20 20 Neofusicoccum parvum (16)
Neopestalotiopsis spp. (11)
Botrytis spp. (5)
Epicoccum spp. (7)		 8 1
4 1
3 1
1 1
2 2017 Srem Šid Duke 20 20 Neofusicoccum parvum (18)
Neopestalotiopsis spp. (14)		 14
11		 2
1
3 2019 Srem Irig Duke 15 5 Lasiodiplodia iraniensis (5) 5 5
4 Kolubara Ub Duke 20 25 Neofusicoccum parvum (18) 6 2
5 2020 Belgrade Sopot Duke 10 8 Neofusicoccum parvum (7)
Alternaria spp. (4)		 4 1
6   Moravica Gornji Milanovac Duke 30 7 Neofusicoccum parvum (5) 4 1
7 2022 Belgrade Slatina Duke 15 20 Neofusicoccum parvum (18)
Diaporthe spp. (12)
Peroneutypa spp. (7)
Fusarium spp. (10)		 4
8
2
2		 1
1
1
2
8 Kolubara Jajčić Duke 30 25 Botryosphaeria dothidea (18)
Diplodia seriata (8)
Diaporthe spp. (5)
Neopestalotiopsis spp. (16)		 12
5
1
4		 4
4
1
3
9 Kolubara Slavkovica Duke 25 23 Botryosphaeria dothidea (15)
Diaporthe spp. (17)		 8
8		 3
2
 
Open in a new tab

* Serial number of the locality as indicated on the map (Figure 1).

Figure 1.

Figure 1

Open in a new tab

Geographic distribution of localities in Serbia included in the survey and detected isolates.

2.2. Morphological and Ecological Characterization

Colony appearance, including colour and shape, was assessed 14 dpi (days post inoculation) on PDA at 24 °C in the dark. Growth rate was determined by measuring two perpendicular colony diameters in five replicates per isolate and calculating an average value for each isolate. To induce sporulation, isolates were cultured on pine needle agar (PNA: 17 g agar, 1 litre distilled H2O and sterilized pine needles placed onto the medium) [38]. The presence and appearance of pycnidia and conidia were observed at 14, 21, 28 and 35 dpi using a compound microscope (Olympus CX41, Olympus Europa SE & Co. KG), and the dimensions of pycnidia and immature and mature conidia were measured (50 and 100 randomly selected, respectively). The selected Lasiodiplodia spp. isolates were physiologically characterized based on colony appearance and ability to grow on PDA at temperatures of 5, 10, 15, 25, 35, 37.5 and 40 °C, as determined by measuring two perpendicular colony diameters in five replicates per isolate and calculating an average value for each temperature. Data were analyzed with SPSS (version 29, IBM, NY, USA) using one-way ANOVA followed by Duncan’s multiple range test at p < 0.05.

2.3. DNA Amplification and Sequencing

Total genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 100 mg of dry mycelium from 7-day-old cultures of 38 selected isolates grown in potato dextrose broth (PDB; 200 g potato, 20 g dextrose and 1 L distilled H2O), following the manufacturer’s instructions. PCR amplification of three genomic regions, including ITS rDNA (38 isolates), TEF1-α (24 isolates) and TUB2 (24 isolates), was performed using the primers ITS1F/ITS4 [39,40], Bt2A/Bt2B [41] and EF1-728/EF1-986 [42], on annealing temperatures 52 °C, 55 °C and 58 °C, respectively. All reactions were performed in a total volume of 25 μL consisting of 12.5 μL of 2× PCR Master Mix (Fermentas, Lithuania), 6.5 μL of RNase-free water, 2.5 μL of both forward and reverse primers (working solution with a final concentration of 100 pmol/μL, Metabion International, Germany) and 1 μL of template DNA. The amplification conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, variable recommended annealing conditions, elongation at 72 °C for 1 min and final elongation for 10 min at 72 °C. The amplicons obtained were stained with ethidium bromide, analyzed by 1% agarose gel electrophoresis and visualized with a UV transilluminator. The PCR products of all genomic regions were sequenced directly in both directions with an automatic sequencer (Automatic Sequencer Macrogen Inc., The Netherlands) using the same primers as for amplification. The consensus sequences were calculated with ClustalW [43], integrated into the software MEGA X [44], and deposited in GenBank (http://www.ncbi.nlm.nih.gov, accessed on 1 April 2025).

2.4. Sequence and Phylogenetic Analyses

Sequences generated from the selected 38 isolates were compared with each other by calculating nucleotide (nt) similarities, as well as with previously deposited isolates available in the GenBank, using the similarity search tool BLAST (version 2.13.0, NCBI) for identification at the genus level.

Multilocus phylogenetic sequence analyses (ITS rDNA, TEF1-α and TUB2) were performed on two data sets, one to clarify the position of 24 Serbian isolates within the family Botryosphaeriaceae and the other to clarify the position of five Lasiodiplodia isolates within the genus Lasiodiplodia. The targeted analyses of Botryosphaeriaceae included 10 previously listed type-derived species (38 reference isolates) and Melanops tulasnei [45,46,47], while other targeted analyses included 34 previously listed type-derived species (49 reference isolates) and Dothiorella viticola [48] as an outgroup (Table 2) with gaps and missing data treated as missing characters. The phylogenetic trees were inferred using the Maximum Likelihood method implemented in MEGA X software [44]. Gamma distributed Tamura-Nei model (G+I) determined by a Model test implemented in MEGA X was used as the best-fitting model of nucleotide substitution. All sites with gaps were omitted. The reliability of the obtained trees was evaluated with 1000 bootstrap replicates.

Table 2.

Isolates of the Botryosphaeriaceae species used in this study.

GenBank Accessions
Species Strain/Isolate Host Country ITS TEF1-α β-Tubulin
Botryosphaeria dothidea CBS115476 Prunus sp. Switzerland AY236949 AY236898 AY236927
Botryosphaeria dothidea CBS110302 Vitis vinifera Portugal AY259092 AY573218 EU673106
Botryosphaeria dothidea CMW44982 Sequoiadendron
giganteum 		Serbia KF575008 KF575040 KF575104
Botryosphaeria dothidea CMW39308 Sequoiadendron
giganteum 		Serbia KF575008 KF575040 KF575104
Botryosphaeria dothidea 34-22-3 Vaccinium corymbosum Serbia PV235336 PV296171 PV278143
Botryosphaeria dothidea 234-22-1 Vaccinium corymbosum Serbia PV268085 PX056801 PX056807
Botryosphaeria dothidea 227-22 Vaccinium corymbosum Serbia PV263064 PX056800 PX056806
Botryosphaeria dothidea 229-22 Vaccinium corymbosum Serbia PV268086 PX056799 PX056805
Botryosphaeria dothidea 224-22-3 Vaccinium corymbosum Serbia PV263065 PX056804 PX056810
Botryosphaeria dothidea 234-22-2 Vaccinium corymbosum Serbia PV263170 PX056802 PX056808
Botryosphaeria dothidea 232-22-2 Vaccinium corymbosum Serbia PX048943 PX056803 PX056809
Botryosphaeria rosaceae CBSCGMCC 3.18007 Malus sp. China KX197074 KX197094 KX197101
Botryosphaeria rosaceae CBSCGMCC 3.18008 Amygdalus sp. China KX197075 KX197095 KX197102
Botryosphaeria rosaceae CFCC 82350 Malus sp. China KX197079 KX197097 KX197106
Botryosphaeria rosaceae CGMCC3.18009 Malus sp. China KX197076 KX197096 KX197103
Botryosphaeria rosaceae CBSCGMCC 3.18010 Pyrus sp. China KX197077 - KX197104
Botryosphaeria rosaceae CBSCGMCC 3.18011 Pyrus sp. China KX197078 - KX197105
Diplodia intemerdia CBS124134 Cydonia sp. Portugal HM036528 GQ923851 KX464798
Diplodia intermedia CBS124462 Malus sylvestris Portugal GQ923858 GQ923826 -
Diplodia sapinea CBS393.84 Pinus nigra Netherlands DQ458895 DQ458880 DQ458863
Diplodia sapinea CBS109725 Pinus patula South Africa DQ458896 DQ458881 DQ458864
Diplodia seriata CMW39384 Thuja occidentalis Serbia DQ458896 DQ458881 DQ458864
Diplodia seriata CMW39376 Chamaecyparis
pisifera 		Serbia KF574996 KF575027 KF575092
Diplodia seriata CBS112555 Vitis vinifera Portugal AY259094 AY573220 DQ458856
Diplodia seriata 224-22-2 Vaccinium corymbosum Serbia PV263172 PV296172 PV278144
Diplodia seriata 224-22-2-1 Vaccinium corymbosum Serbia PX023087 PX056793 PX056796
Diplodia seriata 224-22-2-2 Vaccinium corymbosum Serbia PX022810 PX056794 PX056797
Diplodia seriata 224-22-2-3 Vaccinium corymbosum Serbia PX022815 PX056795 PX056798
Dothiorella viticola CBS 117009 Vitis vinifera Spain AY905554 AY905559 EU673104
Lasiodiplodia americana CERC 1961 Pistacia vera USA: Arizona KP217059 KP217067 KP217075
L. avicenniae CMW 41467 Avicennia marina South Africa KP860835 KP860680 KP860758
L. brasiliense CMM 4015 Mangifera indica Brazil JX464063 JX464049 -
L. bruguierae CMW 41470 Bruguiera gymnorrhiza South Africa NR_147358 KP860678 KP860756
L. citricola CBS 124707 Citrus sp. Iran GU945354 GU945340 KP872405
L. crassispora CBS 118741 Santalum album Australia (WA) DQ103550 EU673303 EU673133
L. crassispora CBS 121770 Acacia mellifera Namibia EU101307 EU101352 -
L. endophytica MFLUCC 18-1 Magnolia candolii China MK501838 MK584572 MK550606
L. egyptiacae CBS 130992 Mangifera indica Egypt JN814397 JN814424 -
L. euphorbicola CMM 3609 Jatropha curcas Brazil KF234543 KF226689 KF254926
L. fujianensis CGMCC: 3.19593 Vaccinium corymbosum China MK802164 OM144905 MK816337
L. gilanensis CBS 124704 Citrus sp. Iran GU945351 GU945342 KP872411
L. gilanensis CBS 128311 Vitis vinifera USA: Missouri HQ288225 HQ288267 -
L. gonubiensis CBS 115812 Syzygium cordatum South Africa AY639595 DQ103566 DQ458860
L. gravistriata CMM 4564 Anacardium humile Brazil KT250949 KT250950 -
L. hormozganensis CBS 124709 Olea sp. Iran GU945355 GU945343 KP872413
L. iraniensis ZLNM3 Mangifera indica Taiwan OR534158 OR552386 OR551924
L. iraniensis ML-1-8-1 Mangifera indica Taiwan OR534131 OR552266 OR551897
L. iraniensis CBS 124710 Salvadora persica Iran GU945346 GU945334 KP872415
L. iraniensis CMM 3610 Jatropha curcas Brazil KF234544 KF226690 KF254927
L. iraniensis 421-19-5 Vaccinium corymbosum Serbia OR856066 PP238619 PP238615
L. iraniensis 421-19-4 Vaccinium corymbosum Serbia OR878143 PP372561 PP238614
L. iraniensis 421-19-3 Vaccinium corymbosum Serbia OR856065 PP238618 PP238613
L. iraniensis 421-19-2 Vaccinium corymbosum Serbia OR856064 PP238617 PP238612
L. iraniensis 421-19 Vaccinium corymbosum Serbia OR727299 PP238616 PP238611
L. laeliocattleyae CBS 167.28 Laeliocattleya sp. Italy KU507487 KU507454 …
L. lignicola MFLUCC 11-0435 On dead wood Thailand JX646797 KU887003 JX646845
L. lignicola CBS 342.78 Sterculia oblonga Germany KX464140 KX464634 KX464908
L. macrospora CMM 3833 Jatropha curcas Brazil KF234557 KF226718 KF254941
L. magnoliae MFLUCC 18-0948 Magnolia candolii China MK499387 MK568537 MK521587
L. mahajangana CBS 124927 Terminalia catappa Madagascar FJ900595 FJ900641 FJ900630
L. mahajangana CMM 1325 Citrus sinensis Brazil KT154760 KT008006 KT154767
L. mahajangana CBS 137785 Retama raetam Tunisia KJ638317 KJ638336 -
L. margaritacea CBS 122519 Adansonia gibbosa Australia (WA) EU144050 EU144065 KX464903
L. mediterranea CBS 137783 Quercus ilex Italy KJ638312 KJ638331 -
L. mitidjana MUM 19.90 Citrus sinensis Algeria: Mitidja MN104115 MN159114 -
L. parva CBS 456.78 Manihot esculenta Colombia EF622083 EF622063 KP872419
L. plurivora CBS 120832 Prunus salicina South Africa EF445362 EF445395 KP872421
L. pontae CMM 1277 Spondias purpurea Brazil KT151794 KT151791 KT151797
L. pseudotheobromae CBS 116459 Gmelina arborea Costa Rica EF622077 EF622057 EU673111
L. pseudotheobromae SEGA21 Vaccinium corymbosum USA JN607093 JN607116 JN607140
L. pseudotheobromae SEGA70 Vaccinium corymbosum USA JN607095 JN607118 JN607142
L. rubropurpurea CBS 118740 Eucalyptus grandis Australia DQ103553 EU673304 EU673136
L. subglobosa CMM 3872 Jatropha curcas Brazil KF234558 KF226721 KF254942
L. thailandica CBS 138760 Mangifera indica Thailand KJ193637 KJ193681 -
L. theobromae CBS 111530 Leucospermum sp. USA: Hawaii EF622074 EF622054 -
L. theobromae CBS 124.13 - USA DQ458890 DQ458875 DQ458858
L. theobromae CBS 164.96 Fruit along coral reef coast Papua New Guinea AY640255 AY640258 EU673110
L. theobromae SEFL3 Vaccinium corymbosum USA JN607091 JN607114 JN607138
L. theobromae SEFL28b Vaccinium corymbosum USA JN607092 JN607115 JN607139
L. vaccinii CGMCC 3.19248 Vaccinium corymbosum China MK157131 MK157158 MK157149
L. venezuelensis CBS 118739 Acacia mangium Venezuela DQ103547 EU673305 EU673129
L. viticola CBS 128313 Vitis vinifera USA: Arkansas HQ288227 HQ288269 HQ288306
L. vitis CBS 124060 Vitis vinifera - KX464148 KX464642 KX464917
Melanops tulasnei CBS116805 Quercus robur Germany FJ824769 KF766423 FJ824780
Neofusicoccum nonquaesitum RGM2880 Vaccinium corymbosum Chile MT790243 MT845319 MT832803
Neofusicoccum nonquaesitum RGM3009 Vaccinium corymbosum Chile MT790223 MT845299 MT832783
Neofusicoccum nonquaesitum RGM2868 Vaccinium corymbosum Chile MT790266 MT845342 MT832826
Neofusicoccum parvum 8-20 Vaccinium corymbosum Serbia OQ31660 OQ342772 OQ473020
Neofusicoccum parvum 3c-20 Vaccinium corymbosum Serbia OQ316605 OQ473018 OQ342770
Neofusicoccum parvum B1-17 Vaccinium corymbosum Serbia OQ316604 OQ342769 OQ473017
Neofusicoccum parvum 4-20 Vaccinium corymbosum Serbia OQ316606 OQ342771 OQ473019
Neofusicoccum parvum 1-21 Vaccinium corymbosum Serbia OQ316608 OQ342773 OQ473021
Neofusicoccum parvum CMW39325 Aesculus
hippocastanum 		Serbia KF575021 KF575045 KF575117
Neofusicoccum parvum CMW39318 Chamaecyparis
lawsoniana 		Serbia KF575022 KF575046 KF575118
Neofusicoccum parvum CBS110301 Vitis vinifera Portugal AY259098 AY573221 EU673095
Neofusicoccum parvum ATCC58191
(CMW9081)		 Populus nigra New Zealand AY236943 AY236888 AY236917
Neofusicoccum parvum 413-19 Vaccinium corymbosum Serbia MW624690 OL456720 OL456719
Neofusicoccum parvum 414-19 Vaccinium corymbosum Serbia MW624691 OL456721 OL415487
Neofusicoccum parvum 790-11 Vaccinium corymbosum Serbia PV235269 PV278148 PV278140
Neofusicoccum parvum 187-17 Vaccinium corymbosum Serbia PV226107 PV278145 PV278139
Neofusicoccum parvum 29-22 Vaccinium corymbosum Serbia PV235282 PV278146 PV278137
Neofusicoccum parvum 30-22 Vaccinium corymbosum Serbia PV235306 PV278147 PV278138
Neofusicoccum parvum RS-BD-1 Vaccinium corymbosum Serbia PV235313 PV278149 PV278141
Neofusicoccum parvum RS-BD-6 Vaccinium corymbosum Serbia PV235322 PV278150 PV278142
Neofusicoccum ribis CBS121.26 Ribes sp. USA AF241177 AY236879 AY236908
Neofusicoccum ribis CBS115475 Ribes sp. USA AY236935 AY236877 AY236906
Open in a new tab

The isolates in bold are obtained in this study.

The position of the Serbian Lasiodiplodia isolates was further evaluated based on the nucleotide polymorphism within the TEF1-α gene. A total of 19 sequences of the closely related L. iraniensis, L. fujianensis, L. thailandica and L. endophytica were aligned and analyzed using the sequence of L. endophytica as a representative [49].

2.5. Pathogenicity Testing

The pathogenicity of 70 Botryosphaeriaceae isolates (40 N. parvum, 20 B. dothidea, 5 D. seriata and 5 L. iraniensis) was tested by the artificial wound inoculation of branches of a 6-year-old healthy blueberry ‘Duke’ from a collection orchard of the Fruit Research Institute Čačak, Serbia, using mycelial plugs, as previously described [16,30,50]. Well-developed, symptomless blueberry branches were superficially sterilized and a clear cut approximately 0.5 cm long incision was made with a sterile scalpel blade without damaging the underlying cambial tissue. Mycelial plugs (5 mm diameter) from the edge of a 4-day-old PDA culture grown at 24 °C were placed under the bark (mycelial surface facing downwards) and the wound was sealed with sterilized moist cotton wool and Parafilm. As a negative control, branches were inoculated with sterile PDA plugs. Three branches were inoculated with each isolate, and the experiment was repeated twice. The pathogenicity of the isolates was assessed 14 dpi. Re-isolations were made from all symptomatic cuttings using the same methods as for isolation.

2.6. Cultivar Susceptibility Testing

In order to assess the susceptibility of blueberry cultivars to infection, a selected L. iraniensis isolate (421-19) was used for the inoculations of branches of six-year-old healthy plants of nine different blueberry cultivars (‘Aurora’, ‘Barbara Ann’, ‘Bluecrop’, ‘Bluejay’, ‘Draper’, ‘Duke’, ‘Huron’, ‘Patriot’ and ‘Spartan’). The experiment was carried out as previously described for the pathogenicity testing. The disease intensity of the nine blueberry cultivars was assessed after 14 dpi. For the purpose of rating, the following 0–4 scale was established in this study based on symptom intensity: 0—no reaction; 1—surface necrosis near the wounded spot; 2—necrosis length from 2 to 20 mm; 3—necrosis length from 21 to 40 mm; and 4—necrosis length greater than 40 mm. The inoculations were performed in 3 replicates and the entire experiment was performed twice. The data were analyzed with the SPSS Software (version 29, IBM, USA) using one-way ANOVA followed by Duncan’s multiple range test at p < 0.05.

3. Results

3.1. Disease Symptoms and Isolates

During the survey, diseased blueberry plants were observed at the nine locations in Serbia (Figure 1), from which 153 samples were collected (Table 1), resulting in 236 isolates, of which representative monosporial isolates were morphologically categorized into 11 morphogroups, of which one or several representative isolates were identified by sequencing the ITS region to the genus level. A total of 128 Botryosphaeriaceae-like isolates were detected in single (three locations) or mixed infection with several non-Botryosphaeriaceae species (Table 1). Symptomatic plants were randomly distributed in groups along the rows or patches of different sizes in the orchards. All sampled orchards were up to six years old and disease incidence was estimated at sampling and ranged between 10 and 30% (mean 20.6%). The plants showed symptoms such as twig dieback, stem blight and wilt, followed by whole plant decay (Figure 2A,G,M and Figure 3A,C). Cross sections of symptomatic branches showed varying degrees of internal tissue necrosis, which correlated with symptom intensity (Figure 2B,H,N and Figure 3B). The spatial distribution of diseased plants along rows or groups of plants in close proximity is probably due to long-distance dispersal by planting material, which is responsible for the introduction of pathogens into the orchards, as well as the spread of the inoculum over short distances within the orchards by the movement of raindrops. Among the Botryosphaeriaceae-like isolates, four species were detected by ITS sequencing. N. parvum was the most prevalent with a detection frequency of 34.75% (82 isolates out of 236) (six out of nine localities). B. dothidea was detected in two localities with a detection frequency of 13.98% (33/236). D. seriata and L. iraniensis were both represented by five isolates from two individual localities with a detection frequency of 6.25 and 3.91, respectively. No species-specific symptomatology was observed. For further detailed characterization, twenty-four isolates were selected, eight isolates of N. parvum, seven of B. dothidea, four of D. seriata and five of L. iraniensis.

Figure 2.

Figure 2

Open in a new tab

Symptomatology and morphology of Botryosphaeriaceae isolates from blueberry in Serbia: stem blight, wilting and inner tissue necrosis caused by Neofusicoccum parvum (A,B), Botryosphaeria dothidea (G,H) and Diplodia seriata (M,N); surface and reverse side of two weeks old colonies on PDA of N. parvum (C,D), B. dothidea (I,J) and D. seriata (O,P); pycnidium and conidia four weeks post inoculation on PNA of N. parvum (E,F), B. dothidea (K,L) and D. seriata (Q,R).

Figure 3.

Figure 3

Open in a new tab

Symptomatology and morphology of Lasiodiplodia iraniensis isolates from Serbia: stem blight and wilting of blueberry (A); inner tissue necrosis (B); numerous pycnidia protruding bark on diseased branches (C); one (D) and two week old colonies on PDA (E); unicellular hyaline immature and pigmented mature conidia four weeks post inoculation on PNA (F); pigmented, 1-septate conidia with longitudinal striations (G), necrosis on inoculated branches of nine different blueberry cultivars: ‘Aurora’, ‘Spartan’, ‘Barbara Ann’, ‘Patriot’, ‘Huron’, ‘Draper’, ‘Bluejay’, ‘Bluecrop’ and ‘Duke’ (H–P).

3.2. Fungal Morphology

The observed morphological characteristics of 70 Botryosphaeriaceae isolates from blueberries in Serbia showed stable uniform morphological characteristics within the four Botryosphaeriaceae species detected.

All 40 N. parvum isolates had a uniform appearance and initially formed white colonies with a grey centre after three days of incubation. With ageing, the colour of the colonies changed in all isolates, so that after seven days they were olive-grey on the surface and greenish grey on the reverse side and finally after two weeks of incubation they were dark grey on the surface and almost black on the reverse side. The aerial mycelium of all isolates was woolly and dense and often grouped in tufts that reached the lid of the Petri dish (Figure 2C,D). All isolates grew fast with average daily growth rates of 14.83 ± 1.09 mm, with no statistical differences between isolates. On PNA, all isolates formed globose blackish pycnidia after two weeks with average dimensions of (250-) 619.80 (-1250) × (200-) 547.75 (-1000) µm (Figure 2E), in which hyaline, fusiform to ellipsoidal, aseptate conidia (16.82–19.06 × 6.54–10.54 µm, average 17.94 ± 1.12 µm × 8.59 ± 2.05 µm) were visible after four weeks of incubation (Figure 2F).

All 20 isolates of B. dothidea also showed a uniform morphology and initially formed white, almost transparent colonies, which became darker in the centre after three days and dark olive-grey after seven days. With ageing after two weeks of incubation, the colonies became dark grey on the surface and dark brown, almost black, on the reverse, all with dense aerial mycelium, and often grouped in tufts that reached the lid of the Petri dish (Figure 2I,J). The average growth rate for all isolates was 13.5 ± 0.92 mm, with no statistical differences. After two weeks of incubation on PNA, all isolates developed blackish pycnidia with an average size of (230-) 365 (-500) × (200-) 287.5 (-375) µm (Figure 2K), with hyaline, fusiform, mostly aseptate conidia (24.91–29.09 × 6.88–9.12 µm, average 27 ± 2.09 × 8 ± 1.12 µm) developed well after four weeks of incubation (Figure 2L).

All five isolates of D. seriata also showed no differences in the appearance of the colonies and initially formed whitish colonies with a visible light olive-brown centre after three days. With further incubation, the colonies became dark olive-grey on the surface and dark grey, almost black, on the reverse after two weeks (Figure 2O,P). The aerial mycelium was dense and fluffy. The colonies of all isolates were fast growing with average growth of 26.2 ± 1.05 mm, overgrowing the entire surface of the Petri dish within two days, with no statistical differences. After two weeks of incubation on PNA, blackish pycnidia (Figure 2Q) could be observed, but they were immersed in the needles, woolly and densely covered with mycelium, making it difficult to determine the exact dimensions. One week after pycnidia formation (three weeks after inoculation on PNA), ovoid to oblong, elliptical, aseptate conidia could be observed (Figure 2R), which were initially hyaline and turned brown with age and were mainly uniseptate (22.95–26.75 × 9.50–10.75 µm, average 25 × 10 ± 1.15 μm), with no statistical differences between the five characterized isolates.

All five isolates of L. iraniensis exhibited uniform morphological characteristics on PDA and formed fast-growing, abundant aerial colonies with an average daily growth rate of 23.6 ± 1.12 mm and overgrew the surface of a 90 mm Petri dish in two days. Initially, the colonies of all isolates were whitish to smoky grey and became grey to olivaceous at the surface and dark, almost black, on the reverse side after two weeks (Figure 3D,E). Sporulation was induced on PNA and blueberry branches, where all isolates formed globose, black pycnidia covered with a dense mycelium (with average dimensions of (520-) 612.5 (-950) × (300-) 400 (-450) µm) after 14 dpi (Figure 3C). The presence of unicellular, hyaline, grey, immature conidia was recorded three weeks after inoculation (19.60–24.40 × 15.0 µm, average 22.00 ± 2.4 × 15.00 ± 0 µm). Approximately 60% of the conidia were pigmented, ellipsoid to ovoid, 1-septate with longitudinal striations (average (20.15–23.35 × 9.75–12.75 µm, 21.75 ± 1.6 × 11.25 ± 1.5 µm) four weeks after inoculation (Figure 3F), and after five weeks, all conidia were mature and pigmented (Figure 3G). There were no statistical differences between the five isolates in terms of growth rate and length of immature versus mature conidia (), except that the immature conidia were wider compared to the mature conidia (Fisher LSD method and 95% confidence).

Ecological characterization of L. iraniensis showed that none of the isolates were able to grow at 5 °C and 40 °C, while growth was recorded at cardinal temperatures of 10 and 37.5 °C (average daily growth for all isolates 6.9 and 2.65 mm, respectively). All isolates grew fastest at 25 and 35 °C (average for all isolates 23.6 and 22.35 mm, respectively). None of the isolates produced pink pigment on PDA at 35 °C in darkness.

3.3. Molecular Identification and Phylogenetic Analyses

BLASTn analyses for each of the ITS, TEF1-α and TUB2 sequences of the morphologically characterized isolates confirmed the identification and proved that eight N. parvum isolates generated in this study share 98.5–100% nucleotide (nt) similarity with N. parvum ex-type isolate CBS 112931, seven isolates of B. dothidea 97.4–100% nt similarity with B. dothidea ex-type isolate CBS 115476 and four isolates of D. seriata 96.1–100% similarity with D. seriata ex-type (CBS 112555). The sequences of five L. iraniensis isolates had 100% nt similarity and 99.6–100% nt sequence similarity to sequences of L. iraniensis (including the ex-type isolate CBS 124710), L. pseudotheobromae, L. theobromae and L. gonubiensis. Similarly, TEF1-α and TUB2 Lasiodiplodia sequence analyses showed that the Serbian isolates have 99.3–99.7% and 97.2–100% nt sequence similarity with sequences of multiple Lasiodiplodia species, respectively.

A multi-locus phylogenetic analysis based on the combined ITS, TEF1-α and TUB2 gene regions using the Maximum likelihood method which included 24 Serbian sequences from four species (N. parvum, B. dothidea, D. seriata, L. iraniensis) and 40 selected isolates from the Botryosphaeriaceae family belonging to 10 species yielded a phylogenetic tree that clearly resolved the topology of several well-supported clades corresponding to N. parvum, B. dothidea, D. seriata, L. iraniensis and other related species. The Serbian isolates clustered within their respective species clades and formed well-supported subclades together with the corresponding ex-type or reference strains (Figure 4). Within the N. parvum clade four subclades are indicated, all comprising 1–4 isolates from Serbia, demonstrating the diversity of blueberry isolates in Serbia.

Figure 4.

Figure 4

Open in a new tab

Maximum likelihood phylogenetic tree inferred from concatenated ITS rDNA, TEF1-α and TUB2 genes of 24 Serbian and 10 previously listed type-derived Botryosphaeriaceae species (38 reference isolates) and Melanops tulasnei as an outgroup. Phylogram was generated with MEGA X using Tamura-Nei model Gamma distributed (G+I) [44]. Bootstrap analysis was performed with 1000 replicates and bootstrap values (>50%) are shown next to relevant branches. The Serbian Botryosphaeriaceae isolates are orange coloured.

Phylogenetic analyses of the ITS, TEF1-α and TUB2 sequences using the Maximum likelihood method, which included five Serbian and fifty selected Lasiodiplodia isolates belonging to 35 species, yielded a phylogenetic tree whose topology and resolution are consistent with previous identification of publicly available isolates (Figure 5). The well-supported branch, which includes the closely related L. iraniensis, L. fujianensis, L. thailandica and L. endophytica, also included all Serbian isolates more closely related to L. iraniensis and L. fujianensis.

Figure 5.

Figure 5

Open in a new tab

Maximum likelihood phylogenetic tree inferred from concatenated ITS rDNA, TEF1-α and TUB2 genes of 5 Serbian and 34 previously listed type-derived species (54 reference isolates) Lasiodiplodia spp. and Dothiorella viticola as an outgroup. Phylogram was generated with MEGA X using Tamura-Nei model Gamma distributed (G+I) [44]. Bootstrap analysis was performed with 1000 replicates and bootstrap values (>70%) are shown next to relevant branches. The Serbian Lasiodiplodia iraniensis isolates are orange coloured.

Subsequent TEF1-α sequence analyses of L. iraniensis, L. fujianensis, L. thailandica and L. endophytica revealed polymorphism at several positions (Table 3). In the analyzed set, L. thailandica isolates were unique as they had adenine at positions 14 and 55 and an insertion at positions 60–67, while all isolates of L. iraniensis, including Serbian isolates, were unique as they had adenine at positions 16 and 68. L. iraniensis could be easily distinguished from the closely related L. fujianensis, which is also a pathogen of blueberries [17] and has cytosine and thymine at positions 16 and 68, respectively. In addition, all 15 L. iraniensis isolates available to date form two separate haplotypes based on single nucleotide polymorphism, as they have either cytosine (9 isolates) or thymine (5 isolates) at position 137.

Table 3.

Translation elongation factor 1α gene nucleotide polymorphism of all available isolates of Lasiodiplodia iraniensis, L. fujianensis, L. thailandica and L. endophytica.

Lasiodiplodia spp. and Accession Number of the Isolate Nucleotide Alignment Using L. endophytica MK584572 as Representative
14 16 55 60–67 68 128 137 159
L.endophytica MK584572 [49] C C G - T C C G
L. thailandica MW183805 [51] A C A insertion T T C G
L. thailandica OQ509100 [52] A C A insertion T C C G
L. thailandica KJ93681 [52] A C A insertion T T C G
L. fujianensis MK887178 [17] C C G - T C C C
L. iraniensis GU945334 [20] C A G - A C T C
L. iraniensis GU945336 [20] C A G - A C T C
L. iraniensis GU945337 [20] C A G - A C T C
L. iraniensis ON975017 [31] C A G - A C T C
L. iraniensis OR114284 [30] C A G - A C T C
L. iraniensis PP389268 [32] - - G - A C C C
L. iraniensis PP389275 [32] - - G - A C C C
L. iraniensis PP389256 [32] - - G - A C C C
L. iraniensis (syn. L. jatrophicola) KF226690 [53] C A G - A C C C
L. iraniensis PP238619 (this study) C A G - A C C C
L. iraniensis PP372561 (this study) C A G - A C C C
L. iraniensis PP238618 (this study) C A G - A C C C
L. iraniensis PP238617 (this study) C A G - A C C C
L. iraniensis PP238616 (this study) C A G - A C C C
Open in a new tab

Different background color indicates differences in nucleotides at particular position.

3.4. Pathogenicity

All 70 detected isolates of Botryosphaeriaceae caused visible symptoms 14 dpi on all inoculated branches, which completely resembled the symptoms of a natural infection. All isolates showed uniform pathogenicity and caused similar reactions in terms of the appearance and intensity of symptoms on the inoculated branches. A large number of pycnidia were observed in the necrotic area of all inoculated branches. No symptoms were observed on the control plants. All isolates were easily reisolated from all inoculated and symptomatic branches, so that Koch’s postulates were fulfilled.

3.5. Cultivar Susceptibility

When evaluating the response of cultivars to inoculation with the selected L iraniensis isolate 421-19, visible symptoms of necrosis were well developed 14 dpi on all inoculated branches of all nine blueberry cultivars tested, namely ‘Aurora’, ‘Barbara Ann’, ‘Bluecrop’, ‘Bluejay’, ‘Draper’, ‘Duke’, ‘Huron’, ‘Patriot’ and ‘Spartan’. The appearance of symptoms was similar in all cultivars and resembled a natural infection, while the control plants of all inoculated cultivars showed no symptoms. The intensity of necrosis varied from cultivar to cultivar (Figure 2H–P), and statistical analysis revealed that symptom development was significantly dependent on cultivar (p < 0.001). The cultivar ‘Duke’ proved to be the most susceptible cultivar with an average score of 3.17 ± 0.983, while the remaining eight cultivars formed a statistically uniform group with similar disease intensity, with average scores of 1.049 ± 0.105 (‘Aurora’)–2.17 ± 0.983 (‘Bluejay’ and ‘Draper’) (Figure 6).

Figure 6.

Figure 6

Open in a new tab

Lasiodiplodia iraniensis: susceptibility of nine blueberry cultivars analyzed with one-way ANOVA followed by Duncan’s multiple range tests at p < 0.05 using SPSS software (IBM, USA) assessed 14 days post inoculation, following 0–4 scale based on the symptom intensity: 0—no reaction; 1—surface necrosis near wounded spot; 2—necrosis lengths from 2 to 20 mm; 3—necrosis lengths from 21 to 40 mm, 4—necrosis length longer than 40 mm. The bars represent standard deviation. Values labelled with the same letter do not differ significantly.

4. Discussion

As a result of our symptom-based study of blueberry dieback in the main growing areas, we found N. parvum, B. dothidea, D. seriata and L. iraniensis, mainly in the form of mixed infections, causing stem blight and plant decay with an average disease incidence of over 20% in all orchards in Serbia. Both D. seriata and L. iraniensis were detected for the first time on blueberries in Serbia, and L. iraniensis was detected for the first time on blueberries worldwide.

N. parvum and B. dothidea are known to have a broad host range [21,52] and were recently detected on blueberries in Serbia [36]. Our study revealed a high prevalence of N. parvum, which is comparable to other blueberry growing areas in the world [8,38,54]. In Serbia, both N. parvum and B. dothidea are known pathogens of trees and shrubs [45,55,56,57], and in addition, B. dothidea is a known post-harvest pathogen of apples and quinces [58,59,60] and a root rot pathogen of sugar beet [47]. Although D. seriata, the third species detected, is known to infect trees and shrubs in Serbia [55] and after harvest on apples and quinces [59,61], it was not known to infect blueberries prior to our study. D. seriata is not very common in blueberries worldwide and has been found in a couple of samples in New Zealand [6,10] and the United States [62], which is similar to the situation in Serbia.

The fourth species detected, L. iraniensis, is a new pathogen for Serbia and was detected in blueberries for the first time worldwide. L. iraniensis has so far been detected mainly in tropical plants and nuts [19,20,22,23,24,25,26,27,28,29,30,31,32]. To date, at least 10 different Lasiodiplodia species have been described as blueberry pathogens. L. chinensis [16], L. clavispora, L. fujianensis, L. henanica, L. nanpingensis [17] and L. pseudotheobromae have been described in China [63], and L. laeliocattleyae in Peru [50]. L. mediterranea has been recorded in the USA [64], Australia [65] and Mexico [66], while L. theobromae occurs in Spain [9,67], China [8], Peru [50], the USA [7] and Australia [65]. L. vaccinii has been recorded in China [16], which shows that this genus could be associated with blueberries in general.

Infected blueberry plants in Serbia showed typical symptoms of Botryosphaeria stem blight [9,11,17,50] and could not be associated with any of the four detected species. Conventional identification of the Serbian isolates based on morphology and growth rate showed that they share characteristics of N. parvum [9,14,68,69,70,71,72], B. dothidea [69,72,73,74,75,76,77], D. seriata [59,61,72,78,79,80,81] and L. iraniensis [19,20,30,32]

Phylogenetic analyses of all detected Botryosphaeriaceae not only confirmed the identity of N. parvum, B. dothidea and D. seriata, but also revealed considerable diversity among isolates of N. parvum in Serbia, which is comparable to the high genetic variation observed in the New Zealand population from grapevines [82] or in Korea on Japanese bay trees [70] as well as in the production of pathogenicity-related toxins in N. parvum populations in France and Portugal [83]. A previous characterization of the population of N. parvum from blueberries in Serbia [36] did not reveal significant diversity among isolates, possibly due to a lower number of sampled orchards where infection could be due to a single introduction. In our study, diversity was shown to be the likely result of multiple introductions. The reverse situation and low diversity within the B. dothidea branch and between isolates from Serbia were similar to the situation of walnut in France [84] and olive in Croatia [72]. The low diversity among isolates of D. seriata detected in our study is to be expected as all isolates originated from a single field, probably as a result of a single introduction, although some variability in the D. serata population has been observed elsewhere [85,86].

The identity of Serbian L. iraniensis within the Botryosphaeriaceae as well as within Lasiodiplodia spp. could not be fully confirmed by phylogenetic analyses based on three loci, as has already been shown for some Lasiodiplodia isolates [52,62]. The Serbian isolates branched with the closely related L. iraniensis, L. fujianensis, L. thailandica and L. endophytica [17,21,49], but also share some of the morphological characteristics such as colony appearance and growth rate [17,19,20,30,32,87]. All four species can be clearly distinguished by the presence and size of the pycnidium as well as the septation and colour of the mature conidia. The Serbian L. iraniensis isolates form pycnidia with an average size of 612.5 µm (up to 850 µm), which is consistent with previously published values for L. iraniensis (up to 980 µm, [20]) and differs markedly from the larger pycnidia of L. fujianensis (up to 1.3 mm, [17]) and the much smaller pycnidia of L. thailandica [87], while L. endophytica does not sporulate in culture [49]. The morphology of the conidia is also a solid tool to distinguish between these four species. Serbian and all previously published isolates of L. iraniensis form pigmented, dark brown, mature conidia that are 1-septate [19,20,31,32,88,89]. The closely related L. fujianensis can be easily distinguished as it forms pigmented but aseptate mature conidia [17], while L. thailandica is characterized by the fact that most mature conidia remain hyaline [49,87,88].

Sequence analysis of the TEF1-α gene provided further confirmation of the clear distinction of L. iraniensis from the closely related L. fujianensis, which was recently detected as a blueberry pathogen in China [17], and from the phylogenetically closely related L. thailandica and L. endophytica, which were not recorded as blueberry pathogens. All available L. iraniensis, including the five Serbian isolates, shared adenine at positions 16 and 68 of the analyzed fragment of the TEF1-α gene, which is a unique sequence feature. In our study, we also found that the previously characterized population of L. iraniensis consists of two haplotypes based on the presence of cytosine or thymine at position 137 of the TEF1-α gene, which represents the first worldwide population analysis of this pathogen. The Serbian isolates belong to the rarer cytosine–haplotype and are identical to the L. iraniensis isolates from Jatropha curcas in Brazil (described as L. jatrophicola, [17,53]) and sweet orange in the USA [32]. The potential role and importance of this diversity in the L. iraniensis population will likely become clearer as additional data and isolates become available and characterized.

There are no studies on the susceptibility of different blueberry accessions or cultivars to Lasiodiplodia spp. Even the data on cultivars naturally infected with Lasiodiplodia spp. are limited. In China, L. theobromae was isolated from the cultivar ‘Misty’ and L. pseudotheobromae from M6 [8]. Our studies on the susceptibility of nine blueberry cultivars are valuable and provide the first data on the presence of different levels of susceptibility in nine tested cultivars. Blueberry ‘Duke’ was found to be significantly more susceptible compared to the other cultivars, which should be further confirmed under different conditions and in other blueberry growing regions. The tested cultivars ‘Aurora’, ‘Bluecrop’ and ‘Bluejay’, which are predominant in blueberry cultivation in the USA [90], responded well and developed low disease severity. The observed difference between the blueberry cultivars tested and the fact that L. iraniensis was isolated from ‘Duke’ in this study may indicate a possible link between natural infection and susceptibility of a particular cultivar.

In Serbia, blueberry ‘Duke’ as the most commonly grown cultivar [3,4], characterized by high susceptibility, is seriously threatened by Bortyosphaeriaceae and especially the emergence of D. seriata and L. iraniensis as new blueberry pathogens. Limiting options for the overall management of Botryosphaeriaceae stem blight diseases emphasize the use of disease-free planting material and the avoidance of injuring plants [11]. In our study, the majority of orchards were in their second or third year of production, meaning that planting material is a likely source of infection, as has been shown previously for many Botryosphaeriaceae [10,21,91]. It would be beneficial for Serbian producers if the control of production and, above all, the import of blueberry planting material in Serbia were strengthened and improved. In view of the fact that the quarantine status of L. pseudotheobromae and L. iraniensis has been discussed [18,19], the standard procedure in the international trade of blueberry planting material should be analyzed and reconsidered. Our results offered a solution as we identified less or moderately susceptible blueberry cultivars to be grown in the affected areas and even more emphasized the need to use pathogen-free planting material in all blueberry-growing areas worldwide.

Abbreviations

The following abbreviations are used in this manuscript:

PDA Potato Dextrose Agar
PNA Pine Needle Agar
PDB Potato Dextrose Broth
ITS Internal Transcribed Spacer
TEF1-α Translation Elongation Factor 1α
TUB2 Beta Tubulin
DNA Deoxyribonucleic Acid
Nt Nucleotide
Bp Base Pair
Dpi Days Per Inoculation
Cv Cultivar
PCR Polymerase Chain Reaction
Open in a new tab

Author Contributions

Conceptualization, A.B., M.M., M.V. and D.J.; methodology, A.B., M.V., M.M., M.G., B.V., D.J. and T.V.; software, M.V. and M.M.; validation, A.B., M.V., M.M., M.G., B.V., D.J. and T.V.; formal analysis, M.V., M.M.; investigation, A.B., M.V., M.M. and M.G.; resources, A.B., M.V., M.M. and D.J.; data curation, M.M. and M.V.; writing—original draft preparation, M.M., M.V., M.G. and A.B.; writing—review and editing, A.B., M.V., M.M., M.G., B.V., D.J. and T.V.; visualization, M.M. and M.V.; supervision, A.B.; project administration, A.B.; funding acquisition, A.B. and M.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data set available on request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by grants 451-03-137/2025-03/200116, 451-03-136/2025-03/200215 and 451-03-137/2025-03/200383 of the Ministry of Science, Technological Development and Innovation of the Republic of Serbia.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

References

  • 1.Retamales J.B., Hancock J.F. Blueberries. 2nd ed. Bell and Bain Ltd.; Glasgow, UK: 2018. pp. 1–323. [Google Scholar]
  • 2.Kalt W., Dufour D. Health functionality of blueberries. HortTechnology. 1997;7:216–221. doi: 10.21273/HORTTECH.7.3.216. [DOI] [Google Scholar]
  • 3.Leposavić A., Jevremović D. Blueberry-Technology of Growing, Protection and Processing. Scientific Pomological Society of Serbia; Belgrade, Serbia: 2020. [Google Scholar]
  • 4.Milivojević J. Jagodaste Voćke. Univerzitet u Beogradu, Poljoprivredni Fakultet; Beograd-Zemun, Serbia: 2022. pp. 339–429. [Google Scholar]
  • 5.Milholland R.D., Galletta G.J. Pathogenic variation among isolates of Botryosphaeria corticis on blueberry. Phytopathology. 1969;59:1540–1543. [Google Scholar]
  • 6.Sammonds J., Billones R., Rocchetti M., Ridgway H.J., Walter M., Jaspers M.V. Survey of blueberry farms for Botryosphaeria dieback and crown rot pathogens. N. Z. Plant Prot. 2009;62:238–242. doi: 10.30843/nzpp.2009.62.4826. [DOI] [Google Scholar]
  • 7.Wright A.F., Harmon P.F. First report of Lasiodiplodia theobromae associated with stem blight of southern highbush blueberries in Florida. Plant Dis. 2009;93:962. doi: 10.1094/PDIS-93-9-0962C. [DOI] [PubMed] [Google Scholar]
  • 8.Xu C., Zhang H., Zhou Z., Hu T., Wang S., Wang Y., Cao K. Identification and distribution of Botryosphaeriaceae species associated with blueberry stem blight in China. Eur. J. Plant Pathol. 2015;143:737–752. doi: 10.1007/s10658-015-0724-6. [DOI] [Google Scholar]
  • 9.Aviles M., De los Santos B., Borrero C. Increase of canker disease severity in blueberries caused by Neofusicoccum parvum or Lasiodiplodia theobromae due to interaction with Macrophomina phaseolina root infection. Eur. J. Plant Pathol. 2021;159:655–663. doi: 10.1007/s10658-020-02195-3. [DOI] [Google Scholar]
  • 10.Tennakoon K.M.S., Ridgway H.J., Jaspers M.V., Jones E.E. Botryosphaeriaceae species associated with blueberry dieback and sources of primary inoculum in propagation nurseries in New Zealand. Eur. J. Plant Pathol. 2017;150:363–374. doi: 10.1007/s10658-017-1283-9. [DOI] [Google Scholar]
  • 11.Ru S., Ding S., Oliver J.E., Amodu A. A review of Botryosphaeria stem blight disease of blueberry from the perspective of plant breeding. Agriculture. 2022;13:100. doi: 10.3390/agriculture13010100. [DOI] [Google Scholar]
  • 12.Garcia J.F., Lawrence D.P., Morales-Cruz A., Travadon R., Minio A., Hernandez-Martinez R., Rolshausen P.E., Baumgartner K., Cantu D. Phylogenomics of plant-associated Botryosphaeriaceae species. Front. Microbiol. 2021;12:652802. doi: 10.3389/fmicb.2021.652802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Denman S., Crous P.W., Taylor J.E., Kang J.C., Pascoe I., Wingfield M.J. An overview of the taxonomic history of Botryosphaeria, and a re-evaluation of its anamorphs based on morphology and ITS rDNA phylogeny. Stud. Mycol. 2000;45:129–140. [Google Scholar]
  • 14.Phillips A.J.L., Alves A., Abdollahzadeh J., Slippers B., Wingfield M.J., Groenewald J.Z., Crous P.W. The Botryosphaeriaceae: Genera and species known from culture. Stud. Mycol. 2013;76:51–67. doi: 10.3114/sim0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Slippers B., Crous P.W., Jami F., Groenewald J.Z., Wingfield M.J. Diversity in the Botryosphaeriales: Looking back, looking forward. Fungal Biol. 2017;121:307–321. doi: 10.1016/j.funbio.2017.02.002. [DOI] [PubMed] [Google Scholar]
  • 16.Zhao L., Wang Y., He W., Zhang Y. Stem blight of blueberry caused by Lasiodiplodia vaccinii sp. nov. in China. Plant Dis. 2019;103:2041–2050. doi: 10.1094/PDIS-01-19-0079-RE. [DOI] [PubMed] [Google Scholar]
  • 17.Wang Y., Zhang Y., Bhoyroo V., Rampadarath S., Jeewon R. Multigene phylogenetics and morphology reveal five novel Lasiodiplodia species associated with blueberries. Life. 2021;11:657. doi: 10.3390/life11070657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.European Food Safety Authority (EFSA) Plant health horizon scanning newsletter. EFSA Support. Publ. 2023;20:7813E. doi: 10.2903/sp.efsa.2023.EN-7813. [DOI] [Google Scholar]
  • 19.Jibrin M.O., Liu Q., Huang Y., Urbina H., Gazis R., Zhang S. Lasiodiplodia iraniensis, a new causal agent of tuber rot on yam (Dioscorea species) imported into the United States and implication for quarantine decisions. Plant Dis. 2022;106:3027–3032. doi: 10.1094/PDIS-11-21-2421-SC. [DOI] [PubMed] [Google Scholar]
  • 20.Abdollahzadeh J., Javadi A., Mohammadi Goltapeh E., Zare R., Phillips A.J.L. Phylogeny and morphology of four new species of Lasiodiplodia from Iran. Persoonia. 2010;25:1–10. doi: 10.3767/003158510X524150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Cruywagen E.M., Slippers B., Roux J., Wingfield M.J. Phylogenetic species recognition and hybridization in Lasiodiplodia: A case study on species from baobabs. Fungal Biol. 2017;121:420–436. doi: 10.1016/j.funbio.2016.07.014. [DOI] [PubMed] [Google Scholar]
  • 22.Rodriguez-Galvez E., Guerrero P., Barradas C., Crous P.W., Alves A. Phylogeny and pathogenicity of Lasiodiplodia species associated with dieback of mango in Peru. Fungal Biol. 2017;121:452–465. doi: 10.1016/j.funbio.2016.06.004. [DOI] [PubMed] [Google Scholar]
  • 23.Bautista-Cruz M.A., Almaguer-Vergas G., Leyva-Mir S.G., Colinas-Leon M.T., Correia K.C., Camacho-Tapia M., Robles-Yerena L., Michereff S.J., Tovar-Pedraza J.M. Phylogeny, distribution, and pathogenicity of Lasiodiplodia species associated with cankers and dieback symptoms of Persian lime in Mexico. Plant Dis. 2019;103:1156–1165. doi: 10.1094/PDIS-06-18-1036-RE. [DOI] [PubMed] [Google Scholar]
  • 24.Sakalidis M.L., Ray J.D., Lanoiselet V., Hardy G.E.S.J., Burgess T.I. Pathogenic Botryosphaeriaceae associated with Mangifera indica in the Kimberley Region of Western Australia. Eur. J. Plant Pathol. 2011;130:379–391. doi: 10.1007/s10658-011-9760-z. [DOI] [Google Scholar]
  • 25.Al-Sadi A.M., Al-Wehaibi A.N., Al-Shariqi R.M., Al-Hammadi M.S., Al-Hosni I.A., Al-Mahmooli I.H., Al-Ghaithi A.G. Population genetic analysis reveals diversity in Lasiodiplodia species infecting date palm, Citrus, and mango in Oman and the UAE. Plant Dis. 2013;97:1363–1369. doi: 10.1094/PDIS-03-13-0245-RE. [DOI] [PubMed] [Google Scholar]
  • 26.Marques M.W., Lima N.B., Morals M.A., Jr., Barbosa M.A.G., Souza B.O., Michereff S., Phillips J.A.L., Câmara M.P.S. Species of Lasiodiplodia associated with mango in Brazil. Fungal Divers. 2013;61:181–193. doi: 10.1007/s13225-013-0231-z. [DOI] [Google Scholar]
  • 27.Li G.Q., Arnold R.J., Liu F.F., Li J.Q., Chen S.F. Identification and pathogenicity of Lasiodiplodia species from Eucalyptus urophylla × grandis, Polyscias balfouriana and Bougainvillea spectabilis in Southern China. J. Phytopathol. 2015;163:956–967. doi: 10.1111/jph.12398. [DOI] [Google Scholar]
  • 28.Netto M.S.B., Lima W.G., Correia K.C., Da Silva C.F.B., Thon M., Marting R.B., Miller R.N.G., Michereff S.J., Câmara M.P.S. Analysis of phylogeny, distribution, and pathogenicity of Botryosphaeriaceae species associated with gummosis of Anacardium in Brazil, with a new species of Lasiodiplodia. Fungal Biol. 2017;121:437–451. doi: 10.1016/j.funbio.2016.07.006. [DOI] [PubMed] [Google Scholar]
  • 29.El-Ganainy S.M., Ismail A.M., Iqbal Z., Elshewy E.S., Alhudaib K.A., Almaghasla M.I., Magista D. Diversity among Lasiodiplodia species causing dieback, root rot and leaf spot on fruit trees in Egypt, and a description of Lasiodiplodia newvalleyensis sp. nov. J. Fungi. 2022;8:1203. doi: 10.3390/jof8111203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Negi N., Krishna R., Meena R.K., Pandey A., Bhandari M.S., Pandey S. First report of Lasiodiplodia iraniensis causing leaf spot disease of Eucalyptus in India. Physiol. Mol. Plant Pathol. 2023;127:102113. doi: 10.1016/j.pmpp.2023.102113. [DOI] [Google Scholar]
  • 31.Silva D.E.M., Vieira R.F.B.S., Inokuti E.M., Almeida M.M.M., Cordeiro M.V.M., Lima C.S., Oster A.H., Silva C.F.B. First report of Lasiodiplodia iraniensis causing crown rot on banana fruits in Brazil. Plant Dis. 2023;107:3315. doi: 10.1094/PDIS-10-22-2361-PDN. [DOI] [Google Scholar]
  • 32.Piattino V., Aiello D., Dardani G., Martino I., Flores M., Aćimović S.G., Spadaro D., Polizzi G., Guarnaccia V. Lasiodiplodia iraniensis and Diaporthe spp. are associated with twig dieback and fruit stem-end rot of sweet orange, Citrus sinensis, in Florida. Horticulturae. 2024;10:406. doi: 10.3390/horticulturae10040406. [DOI] [Google Scholar]
  • 33.Popović T., Blagojević J., Aleksić G., Jelušić A., Krnjajić S., Milovanović P. A blight disease on highbush blueberry associated with Macrophomina phaseolina in Serbia. Can. J. Plant Pathol. 2018;40:121–127. doi: 10.1080/07060661.2017.1415977. [DOI] [Google Scholar]
  • 34.Ristić D., Vučurović I., Živković S., Starović M., Delibašić G., Tanović B., Aleksić G. Fusarium sporotrichioides—Novi patogen borovnice u Srbiji; Proceedings of the XVI Simpozijum o Zaštiti Bilja u Bosni i Hercegovini; Sarajevo, Bosnia and Herzegovina. 5–7 November 2019; pp. 42–43. [Google Scholar]
  • 35.Jevremović D., Vasić T., Živković S., Vasilijević B., Marić M., Vojvodić M., Bulajić A. Neopestalotiopsis clavispora: A causal agent of twig dieback on highbush blueberries in Serbia. J. Plant Dis. Prot. 2022;129:1277–1283. doi: 10.1007/s41348-022-00610-x. [DOI] [Google Scholar]
  • 36.Blagojević J., Aleksić G., Vučurović I., Starović M., Ristić D. Exploring the phylogenetic diversity of Botryosphaeriaceae and Diaporthe species causing dieback and shoot blight of blueberry in Serbia. Phytopathology. 2024;114:1333–1345. doi: 10.1094/PHYTO-04-23-0133-R. [DOI] [PubMed] [Google Scholar]
  • 37.Dhingra O., Sinclair J. Basic Plant Pathology Methods. 2nd ed. CRC Press; Boca Raton, FL, USA: 1995. [DOI] [Google Scholar]
  • 38.Espinoza J.G., Briceño E.X., Chávez E.R., Úrbez-Torres J.R., Latorre B.A. Neofusicoccum spp. associated with stem canker and dieback of blueberry in Chile. Plant Dis. 2009;93:1187–1194. doi: 10.1094/PDIS-93-11-1187. [DOI] [PubMed] [Google Scholar]
  • 39.Gardes M., Bruns T.D. ITS primers with enhanced specificity for basidiomycetes—Application to the identification of mycorrhizae and rusts. Mol. Ecol. 1993;2:113–118. doi: 10.1111/j.1365-294X.1993.tb00005.x. [DOI] [PubMed] [Google Scholar]
  • 40.White T.J., Bruns T., Lee S., Taylor J.W. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M.A., Gelfand D.H., Sninsky J.J., White T.J., editors. PCR Protocols: A Guide to Methods and Applications. Academic Press; New York, NY, USA: 1990. pp. 315–322. [DOI] [Google Scholar]
  • 41.Glass N.L., Donaldson G.C. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 1995;61:1323–1330. doi: 10.1128/aem.61.4.1323-1330.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Carbone I., Kohn L.M. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia. 1999;91:553–556. doi: 10.1080/00275514.1999.12061051. [DOI] [Google Scholar]
  • 43.Thompson J.D., Higgins D.G., Gibson T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Kumar S., Stecher G., Li M., Knyaz C., Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018;35:1547–1549. doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Zlatković M., Keča N., Wingfield M.J., Jami F., Slippers B. Shot hole disease on Prunus laurocerasus caused by Neofusicoccum parvum. Antonie Leeuwenhoek. 2016;109:543–564. doi: 10.1007/s10482-016-0659-8. [DOI] [PubMed] [Google Scholar]
  • 46.Zhou Y.P., Zhang M., Dou Z.P., Zhang Y. Botryosphaeria rosaceae sp. nov. and B. ramosa, new botryosphaeriaceous taxa from China. Mycosphere. 2017;8:162–171. doi: 10.5943/mycosphere/8/2/2. [DOI] [Google Scholar]
  • 47.Vučković N., Duduk N., Rekanović E., Duduk B., Vico I. First report of Botryosphaeria dothidea causing root rot of sugar beet in Serbia. Plant Dis. 2024;108:3658. doi: 10.1094/PDIS-06-24-1275-PDN. [DOI] [Google Scholar]
  • 48.Bezerra J.D.P., Crous P.W., Aiello D., Gullino M.L., Polizzi G., Guarnaccia V. Genetic diversity and pathogenicity of Botryosphaeriaceae species associated with symptomatic citrus plants in Europe. Plants. 2021;10:492. doi: 10.3390/plants10030492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.de Silva N.I., Phillips A.J.L., Liu J.K., Lumyong S., Hyde K.D. Phylogeny and morphology of Lasiodiplodia species associated with Magnolia forest plants. [(accessed on 23 September 2024)];Sci. Rep. 2019 9:14355. doi: 10.1038/s41598-019-50804-x. Available online: https://www.nature.com/articles/s41598-019-50804-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Rodriguez-Galvez E., Hilário S., Lopes A., Alves A. Diversity and pathogenicity of Lasiodiplodia and Neopestalotiopsis species associated with stem blight and dieback of blueberry plants in Peru. Eur. J. Plant Pathol. 2020;157:89–102. doi: 10.1007/s10658-020-01983-1. [DOI] [Google Scholar]
  • 51.Tennakoon D.S., Kuo C.H., Maharachchikumbura S.S.N., Thambugala K.M., Gentekaki E., Phillips A.J.L., Bhat D.J., Wanasinghe D.N., De Silva N.I., Promputtha I., et al. Taxonomic and phylogenetic contributions to Celtis formosana, Ficus ampelas, F. septica, Macaranga tanarius and Morus australis leaf litter inhabiting microfungi. Fungal Divers. 2021;108:1–215. doi: 10.1007/s13225-021-00474-w. [DOI] [Google Scholar]
  • 52.Rathnayaka A.R., Chethana K.W.T., Manawasinghe I.S., Wijesinghe S.N., De Silva N.I., Tennakoon D.S., Phillips A.J.L., Liu J.K., Jones E.B.G., Wang Y., et al. Lasiodiplodia: Generic revision by providing molecular markers, geographical distribution and haplotype diversity. Mycosphere. 2023;14:1254–1339. doi: 10.5943/mycosphere/14/1/14. [DOI] [Google Scholar]
  • 53.Machado A., Pinho D., Pereira O. Phylogeny, identification and pathogenicity of the Botryosphaeriaceae associated with collar and root rot of the biofuel plant Jatropha curcas in Brazil, with a description of new species of Lasiodiplodia. Fungal Divers. 2014;67:231–247. doi: 10.1007/s13225-013-0274-1. [DOI] [Google Scholar]
  • 54.Hilario S., Lopes A., Santos L., Alves A. Botryosphaeriaceae species associated with blueberry stem blight and dieback in the Centre region of Portugal. Eur. J. Plant Pathol. 2020;156:31–44. doi: 10.1007/s10658-019-01860-6. [DOI] [Google Scholar]
  • 55.Zlatković M., Keča N., Wingfield M.J., Jami F., Slippers B. Botryosphaeriaceae associated with the die-back of ornamental trees in Serbia. Forest Pathol. 2016;46:267–282. doi: 10.1007/s10482-016-0659-8. [DOI] [PubMed] [Google Scholar]
  • 56.Zlatković M., Wingfield M.J., Jami F., Slippers B. Genetic uniformity characterizes the Invasive spread of Neofusicoccum parvum. Plant Pathol. 2019;68:1153–1163. doi: 10.1111/ppa.13090. [DOI] [Google Scholar]
  • 57.Karličić V., Jovičić-Petrović J., Marojević V., Zlatković M., Orlović S., Raičević V. Potential of Trichoderma spp. and Pinus sylvestris bark extracts as biocontrol agents against fungal pathogens residing in the Botryosphaeriales. Environ. Sci. Proc. 2021;3:99. doi: 10.3390/IECF2020-07960. [DOI] [Google Scholar]
  • 58.Vasić M., Duduk N., Vico I., Ivanović M.S. First report of Botryosphaeria dothidea causing white rot of apple fruit in Serbia. Plant Dis. 2013;97:1659. doi: 10.1094/PDIS-05-13-0493-PDN. [DOI] [PubMed] [Google Scholar]
  • 59.Vučković N., Vico I., Duduk B., Duduk N. Diversity of Botryosphaeriaceae and Diaporthe species associated with postharvest apple fruit decay in Serbia. Phytopathology. 2022;112:929–943. doi: 10.1094/PHYTO-07-21-0304-R. [DOI] [PubMed] [Google Scholar]
  • 60.Vučković N., Vico I., Duduk N. First report of Botryosphaeria dothidea causing postharvest rot of quince fruits in Serbia. J. Plant Pathol. 2023;105:605. doi: 10.1007/s42161-023-01314-3. [DOI] [Google Scholar]
  • 61.Vico I., Žebeljan A., Vučković N., Vasić M., Duduk N. First report of Diplodia seriata causing postharvest rot of quince fruit in Serbia. Plant Dis. 2017;101:1823. doi: 10.1094/PDIS-04-17-0484-PDN. [DOI] [Google Scholar]
  • 62.Flor N.C., Wright A.F., Huguet-Tapia J., Harmon P.F., Liberti D. Identification of fungi in the Botryosphaeriaceae family associated with stem blight of Vaccinium spp. in the southeastern United States. Fungal Biol. 2022;126:342–355. doi: 10.1016/j.funbio.2022.03.004. [DOI] [PubMed] [Google Scholar]
  • 63.Wang J.N., Zhao H.H., Yu Y.Y., Li X.D., Liang C., Li B.D. The pathogen causing Lasiodiplodia twig blight of blueberry. Mycosystema. 2016;35:657–665. doi: 10.13346/j.mycosystema.150165. [DOI] [Google Scholar]
  • 64.Wiseman M.S., Serdani M., Putnam M.L. A new cane dieback disease of northern highbush blueberry in the United States caused by Lasiodiplodia mediterranea. Plant Dis. 2017;101:1317. doi: 10.1094/PDIS-01-17-0028-PDN. [DOI] [Google Scholar]
  • 65.Scarlett K.A., Shuttleworth L.A., Collins D., Rothwell C.T., Guest D.I., Daniel R. Botryosphaeriales associated with stem blight and dieback of blueberry (Vaccinium spp.) in New South Wales and Western Australia. Australas. Plant Pathol. 2019;48:45–57. doi: 10.1007/s13313-018-0584-6. [DOI] [Google Scholar]
  • 66.Rebollar-Alviter A., Boyzo-Marín J., Silva-Rojas H.V., Ramírez G. Fungi and oomycete pathogens causing stem blight and root rots on blueberry in Central Mexico. Phytopathology. 2013;103:119–120. [Google Scholar]
  • 67.Borrero C., Perez S., Aviles M. First report of canker disease caused by Lasiodiplodia theobromae on blueberry bushes in Spain. Plant Dis. 2019;103:10. doi: 10.1094/PDIS-03-19-0473-PDN. [DOI] [PubMed] [Google Scholar]
  • 68.Latorre B.A., Díaz G.A., Reed M.P. Effect of water activity on in vitro mycelial growth of Neofusicoccum spp. infecting blueberry. Cienc. Investig. Agrar. 2012;9:221–228. doi: 10.4067/S0718-16202012000100019. [DOI] [Google Scholar]
  • 69.Babiker E.M., Stringer S.J., Sakhanokho H.F., Smith B.J. Characterization and pathogenicity of stem blight complex isolates associated with stem blight disease on Vaccinium species. HortScience. 2019;54:1199–1203. doi: 10.21273/HORTSCI14033-19. [DOI] [Google Scholar]
  • 70.Choi S., Paul N.C., Lee K.-H., Kim H.-J., Sang H.M. Morphology, molecular phylogeny, and pathogenicity of Neofusicoccum parvum, associated with leaf spot disease of a new host, the Japanese bay tree (Machilus thunbergii) Forests. 2021;12:440. doi: 10.3390/f12040440. [DOI] [Google Scholar]
  • 71.Milas P., Barra-Bucarei L., Castro J.F., Carrasco-Fernández J., Chilian J., Tapia E., Santelices C., Cisterna-Oyarce V., Muñoz V., Ortiz-Campos J., et al. Identification and distribution of species of Neofusicoccum that cause blueberry stem blight in Chile. Mycologia. 2023;115:326–339. doi: 10.1080/00275514.2023.2180976. [DOI] [PubMed] [Google Scholar]
  • 72.Petrović E., Vrandečić K., Belušić Vozila A., Ćosić J., Godena S. Diversity and pathogenicity of Botryosphaeriaceae species isolated from olives in Istria, Croatia, and evaluation of varietal resistance. Plants. 2024;13:1813. doi: 10.3390/plants13131813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 73.Li X., Yan J., Kong F., Qiao G., Zhang Z., Wang Z. Botryosphaeria dothidea causing canker of grapevine newly reported in China. Plant Pathol. 2010;59:1170. doi: 10.1111/j.1365-3059.2010.02356.x. [DOI] [Google Scholar]
  • 74.Yu L., Rarisara I., Xu S.G., Wu X., Zhao J.R. First report of stem blight of blueberry caused by Botryosphaeria dothidea in China. Plant Dis. 2012;96:1697. doi: 10.1094/PDIS-05-12-0500-PDN. [DOI] [PubMed] [Google Scholar]
  • 75.Yan J., Xie Y., Yao S., Wang Z., Li X. Characterization of Botryosphaeria dothidea, the causal agent of grapevine canker in China. Australas. Plant Pathol. 2012;41:351–357. doi: 10.1007/s13313-012-0135-5. [DOI] [Google Scholar]
  • 76.Marsberg A., Kemler M., Jami F., Nagel J.H., Postma-Smidt A., Naidoo S., Wingfield M.J., Crous P.W., Spatafora J.W., Hesse C.N., et al. Botryosphaeria dothidea: A latent pathogen of global importance to woody plant health. Mol. Plant Pathol. 2017;18:477–488. doi: 10.1111/mpp.12495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 77.Kovač M., Diminić D., Orlović S., Zlatković M. Botryosphaeria Dothidea and Neofusicoccum Yunnanense Causing Canker and Die-Back of Sequoiadendron Giganteum in Croatia. Forests. 2021;12:695. doi: 10.3390/f12060695. [DOI] [Google Scholar]
  • 78.Phillips A.J.L., Crous P.W., Alves A. Diplodia seriata, the anamorph of “Botryosphaeria” obtusa. Fungal Divers. 2007;25:141–155. [Google Scholar]
  • 79.Gonzalez-Dominguez E., Alves A., León M., Armengol J. Characterization of Botryosphaeriaceae species associated with diseased loquat (Eriobotrya japonica) in Spain. Plant Pathol. 2017;66:77–89. doi: 10.1111/ppa.12556. [DOI] [Google Scholar]
  • 80.Arrigoni E., Oliveira Longa C.M., Angeli D., Soini M., Pertot I., Perazzolli M. A fast and reliable method for Diplodia seriata inoculation of trunks and assessment of fungicide efficacy on potted apple plants under greenhouse conditions. Phytopathol. Mediterr. 2019;58:163–173. doi: 10.14601/Phytopathol_Mediterr-23764. [DOI] [Google Scholar]
  • 81.Manetti G., Brunetti A., Sciarroni L., Lumia V., Bechini S., Marangi P., Reverberi M., Scortichini M., Pilotti M. Diplodia seriata Isolated from Declining Olive Trees in Salento (Apulia, Italy): Pathogenicity Trials Give a Glimpse That It Is More Virulent to Drought-Stressed Olive Trees and in a Warmth-Conditioned Environment. Plants. 2024;13:2245. doi: 10.3390/plants13162245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82.Baskarathevan J., Jaspers M.V., Jones E.E., Cruickshank R.H., Ridgway H.J. Genetic and pathogenic diversity of Neofusicoccum parvum in New Zealand vineyards. Fungal Biol. 2012;116:276–288. doi: 10.1016/j.funbio.2011.11.010. [DOI] [PubMed] [Google Scholar]
  • 83.Trotel-Aziz P., Robert-Siegwald G., Fernandez O., Leal C., Villaume S., Guise J.F., Abou-Mansour E., Lebrun M.H., Fontaine F. Diversity of Neofusicoccum parvum for the production of the phytotoxic metabolites (-)-Terremutin and (R)-Mellein. J. Fungi. 2022;8:319. doi: 10.3390/jof8030319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84.Belair M., Picot A., Lepais O., Masson C., Hébrard M., Moronvalle A., Comont G., Gabri Martin V.M., Tréguer S., Laloum Y., et al. Genetic diversity and population structure of Botryosphaeria dothidea and Neofusicoccum parvum on English walnut (Juglans regia L.) in France. Sci. Rep. 2024;14:19817. doi: 10.1038/s41598-024-67613-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Elena G., Garcia-Figueres F., Reigada S., Luque J. Intraspecific variation in Diplodia seriata isolates occurring on grapevines in Spain. Plant Pathol. 2015;64:680–689. doi: 10.1111/ppa.12296. [DOI] [Google Scholar]
  • 86.Bhat A.H., Shah M.D., Padder B.A., Shah Z.A., Dar E.A., Fayaz U., Nain M.S., Ali M.A., Al-Hemaid F.M., Stępień P., et al. Morphological, pathogenic and genetic diversity in Diplodia seriata associated with black rot canker of apple in India. Sci. Rep. 2023;13:15682. doi: 10.1038/s41598-023-42534-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Trakunyingcharoen T., Lombard L., Groenewald J.Z., Cheewangkoon R., To-anun C., Crous P.W. Caulicolous Botryosphaeriales from Thailand. Persoonia. 2015;34:87–99. doi: 10.3767/003158515X685841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88.Alves A., Crous P.W., Correia A., Phillips A.J.L. Morphological and molecular data reveal cryptic speciation in Lasiodiplodia theobromae. Fungal Divers. 2008;28:1–13. [Google Scholar]
  • 89.Dou Z.P., He W., Zhang Y. Does morphology matter in taxonomy of Lasiodiplodia? An answer from Lasiodiplodia hyalina sp. nov. Mycosphere. 2017;8:1014–1027. doi: 10.5943/mycosphere/8/2/5. [DOI] [Google Scholar]
  • 90.Gallardo R.K., Zhang Q., Dossett M., Polashock J.J., Rodriguez-Saona C., Vorsa N., Edger P.P., Ashrafi H., Babiker E., Finn C.E., et al. Breeding trait priorities of the blueberry industry in the United States and Canada. HortScience. 2018;53:1021–1028. doi: 10.21273/HORTSCI12964-18. [DOI] [Google Scholar]
  • 91.Sakalidis M.L., Hardy G.E.S.J., Burgess T.I. Endophytes as potential pathogens of the baobab species Adansonia gregorii: A focus on the Botryosphaeriaceae. Fungal Ecol. 2011;4:1–14. doi: 10.1016/j.funeco.2010.06.001. [DOI] [Google Scholar]

Associated Data

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

Data Availability Statement

Data set available on request from the authors.

Articles from Journal of Fungi are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES