Alternaria yunnanensis sp. nov., a New Alternaria Species Causing Foliage Spot of Rubber Tree in China

Abstract

A new species of Alternaria causing leaf spots on the rubber tree (Hevea brasiliensis) in Yunnan, China, was isolated, examined, and illustrated. Morphologically, it belongs to the section Porri of Alternaria, which produces relatively large conidia and a simple or branched, filamentous long beak. It is, however, characterized by conidiophores gradually enlarging near the apex into a clavate conidiogenous cell and long ellipsoid to obclavate, smooth-walled conidia with a long filamentous beak. Molecular phylogenetic analyses based on ITS rDNA, GAPDH, and TEF1-alpha sequences demonstrate that the phytopathogen falls in the clade of the section Porri, being most closely related to A. sidae, A. sennae, A. deseriticola, A. cyamopsidis, A. rostellata, A. nitrimali, A. crassa, and A. thunbergiae.

1. Introduction

The Alternaria is saprobic and ubiquitous, and some species are well-known as major plant pathogens. They can also cause many human health disorders, while many can play an important role in decay and decomposition [1]. Alternaria was divided into 24 sections on the basis of morphological and multi-loci molecular phylogeny (18S, ITS and 28S rDNA, GAPDH, RPB2, and TEF1-alpha) [2–5]. The section Porri is the largest Alternaria section with species characterized by medium to large conidia with a simple or branched, filamentous long beak [2,3]. This section includes some important phytopathogens, such as A. porri, A. bataticola, A. solani, and A. tomatophila. A. porri causes purple blotch of onion [6], A. bataticola causes leaf petiole and stem blight of sweet potato [7], A. solani is the causative agent of early blight of potato [8], and A. tomatophila is known for causing early blight of tomato [9].

Earlier Martin [10] reported Alternaria sp. infection on Hevea brasiliensis in Mexico. In 2006, another Alternaria leaf blight with similar symptom on rubber trees caused by A. alternata was identified by Roy et al. [11] in India. Our research group reported that A. heveae and A. alternata were able to incite black leaf spot of rubber tree in China [12,13]. The disease symptoms initially appear as minute spots on leaves. The spots are circular with black center surrounded by a yellow halo, and lesions slightly sunken.

During a survey on rubber tree diseases, a fungus isolated from rubber tree leaf spot lesions was morphologically identified to be an Alternaria species in the section Porri but which did not fit any known species of this genus and further confirmed molecularly by grouping within the section of Porri. Herein, we propose this new species with morphological description, illustration, and molecular phylogeny.

2. Materials and methods

2.1. Isolation and cultures

Fungus-infected leaves of the rubber tree were collected in May 2014 from Hula village, Ruili city, Yunnan Province, China. Segmented leaf lesions between symptomatic and healthy tissue were surface sterilized with 70% ethanol for 2 min and then air-dried, plated on potato dextrose agar (PDA; 20 g white potato boiled and filtered, 20 g dextrose, 15 g agar, and 1 L distilled water), and incubated at 28 °C with natural day/night cycles of light for 5–7 days. Single spores were isolated by following the method of Ho and Ko [14] and cultured and transferred onto a new PDA plate to establish pure cultures.

Isolates were maintained at the Herbarium of Department of Plant Pathology (YITC 5109 and YITC 5114), Yunnan institute of Tropic Crops. Ex-type was stored in the Agricultural Culture Collection of China, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (ACCC 39327) and also in the China General Microbial Culture Collection, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (CGMCC3.1890). The type was deposited in the Herbarium Mycologicum Academiae Sinicae, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS247784).

2.2. Pathogenicity assay

Pathogenicity assays using a field rubber tree inoculation method were conducted as previously described by Cai et al. [13]. Fungal conidia were harvested by flooding 7-day-old cultures with sterile water from single conidial cultures, centrifuging, and adjusting the concentration to 1 × 10⁴ conidia/ml, and spraying the spore suspension to the copper-color leaves (approximately 5–7 cm in length) of rubber tree using manual pressure sprayer. Rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags for 48 h. The plastic bags were removed after 2 days postinoculation and monitored daily for symptom development.

2.3. Morphological observations

The isolate YITC 5109 was used for morphological descriptions. Colonial characteristics (color, size, and texture) were assessed after 7 days of growth on the PDA plates and V-8 juice agar (V-8; 100 mL V-8 juice, 0.2 g CaCO₃, 20 g agar, and 900 mL distilled water) at 25 °C under a fluorescent light/dark cycle of 8/16 h. Conidial morphology was examined according to the method of Simmons [15,16]. Isolates were inoculated on potato carrot agar (PCA; 20 g white potato boiled and filtered, 20 g carrot, and 20 g agar in 1 L distilled water) and V-8 juice agar. The plates were stored at 22 °C for 7 days in a chamber without humidity control (a gradually drying atmosphere in unsealed plates). A total of 100 conidia were randomly chosen and measured by using an OLYMPUS CH30RF200 light microscope (Olympus, Tokyo, Japan) with a Moticam 5000 digital camera (Motic Deutschland GmbH, Wetzlar, Germany).

2.4. DNA extraction, amplification and sequencing

Isolates were grown on PDA for 5–7 days and then collected for DNA extraction. Genomic DNA was extracted following a previously described method [17] with some modifications. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region was amplified with the universal primers ITS1 and ITS4 [18], the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) with the primers gpd1 and gpd2 [19], the TEF1-alpha gene (TEF1) with the primers EF1-728F and EF1-986R [20]. The PCR mixtures included 9.5 μL of ddH₂O, 2 μL of each primer (10 μM), 1 μL of genomic DNA, 12.5 μL of 2 × EsTaq Master Mix containing 0.5 units of EsTaq DNA Polymerase, 3 mM of MgCl₂ and 200 µM of each dNTP and 1× PCR buffer (Cwbio, Beijing, China). The PCR reactions were carried out in a Veriti 96-well thermo cycler (Applied Biosystems, Carlsbad, CA). The cycling program was initiated at 94 °C for 5 min, and then followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 58 °C for 30 sec, and elongation at 72 °C for 30 sec, and finalized with an extension at 72 °C for 10 min. PCR products were sequenced by BGI (Beijing Genomics Institute, Beijing, China). The obtained sequences (ITS, GAPDH, and TEF1) were deposited in GenBank with the accession numbers shown in Table 1.

Table 1.

Isolates used in this study and their GenBank accession numbers.

Name	Old name	Strain numbera	Host/Substrate	Locality	GenBank accession numbers
					ITS	GAPDH	TEF1
Alternaria acalyphicola		CBS 541.94; E.G.S. 38.100	Acalypha indica	Seychelles	KJ718097	KJ717952	KJ718446
Alternaria agerati		CBS 117221; E.G.S. 30.001	Ageratum houstonianum	USA, Illinois	KJ718098	KJ717953	KJ718447
Alternaria agripestis		CBS 577.94; E.G.S. 41.034	Euphorbia esula, stem lesion	Canada, Saskatchewan	KJ718099	JQ646356	KJ718448
Alternaria allii	Alternaria porri	CBS 107.28; E.G.S. 48.084	Allium cepa, leaf spot	Puerto Rico	KJ718100	KJ717954	KJ718449
	Alternaria vanuatuensis	CBS 121345; E.G.S. 45.018	Allium cepa, leaf	Vanuatu	KJ718104	KJ717958	KJ718453
Alternaria alternariacida sp. nov.	Alternaria solani	CBS 105.51; ATCC 11078;	Solanum lycopersicum, fruit	UK, England	KJ718105	KJ717959	KJ718454
Alternaria anagallidis		CBS 101004	Anagallis arvensis, leaf spot	New Zealand, Auckland	KJ718107	KJ717960	KJ718455
		CBS 117128; E.G.S. 42.074	Anagallis arvensis, leaf spot	New Zealand, Auckland	KJ718108	KJ717961	KJ718456
Alternaria anodae		PPRI 12376	Anoda cristata, leaf	South Africa, Gauteng	KJ718110	KJ717963	KJ718458
Alternaria aragakii		CBS 594.93; E.G.S. 29.016	Passiflora edulis	USA, Hawaii	KJ718111	KJ717964	KJ718459
Alternaria argyroxiphii		CBS 117222; E.G.S. 35.122	Argyroxiphium sp.	USA, Hawaii	KJ718112	JQ646350	KJ718460
		PPRI 11848	Ipomoea batatas, stem lesion	South Africa, Gauteng	KJ718113	KJ717965	KJ718461
		PPRI 11971	Ipomoea batatas, leaf	South Africa, pumalanga	KJ718114	KJ717966	KJ718462
Alternaria azadirachtae		CBS 116444; E.G.S. 46.195;	Azadirachta indica, leaf spot	Australia, Queensland	KJ718115	KJ717967	KJ718463
		CBS 116445; E.G.S. 46.196;	Azadirachta indica, leaf spot	Australia, Queensland	KJ718116	KJ717968	KJ718464
A. yunnanensis sp. nov.		YITC 5109	Hevea brasiliensis, leaf spot	China,	MH044725	MH044727	MH044726
		YITC 5114	Hevea brasiliensis, leaf spot	China,	MH044728	MH044730	MH044729
Alternaria bataticola		CBS 531.63; IFO 6187	Ipomoea batatas	Japan	KJ718117	JQ646349	KJ718465
		CBS 117096; E.G.S. 42.158;	Ipomoea batatas, leaf spot	Australia, Queensland	KJ718120	KJ717971	KJ718468
Alternaria blumeae	Alternaria brasiliensis	CBS 117215; E.G.S. 39.116	Phaseolus vulgaris, leaf spot	Brazil, Esperito Santo	KJ718125	KJ717976	KJ718473
		CBS 117364; E.G.S. 40.149	Blumea aurita	Thailand, Yala Province	KJ718126	AY562405	KJ718474
Alternaria calendulae		CBS 224.76; ATCC 38903;	Calendula officinalis, leaf spot	Germany	KJ718127	KJ717977	KJ718475
		CBS 116439; E.G.S. 42.197	Rosa sp., leaf spot	New Zealand, Auckland	KJ718129	KJ717979	KJ718477
Alternaria carthami		CBS 635.80	Carthamus tinctorius, leaf	Italy, Perugia	KJ718131	KJ717981	KJ718479
	Alternaria heliophytonis	CBS 116440; E.G.S. 3.143;	Helianthus annuus, leaf	Canada, Saskatchewan	KJ718132	KJ717982	KJ718480
Alternaria carthamicola	Alternaria carthami	CBS 117092; E.G.S. 37.057	Carthamus tinctorius	Iraq	KJ718134	KJ717984	KJ718482
Alternaria cassiae		CBS 478.81; E.G.S. 33.147	Senna obtusifolia, seedling	USA, Mississippi	KJ718135	KJ717985	KJ718483
	Alternaria sauropodis	CBS 116119; E.G.S. 47.112	Sauropus androgynus	Malaysia, Sarawak	KJ718136	KJ717986	KJ718484
	Alternaria hibiscinficiens	CBS 117369; E.G.S. 50.166	Hibiscus sabdariffa, leaf	Fiji	KJ718138	KJ717988	KJ718486
Alternaria catananches sp. nov.		CBS 137456; PD 013/05703936	Catananche caerulea	Netherlands	KJ718139	KJ717989	KJ718487
Alternaria centaureae		CBS 116446; E.G.S. 47.119	Centaurea solstitialis, leaf spot	USA, California	KJ718140	KJ717990	KJ718488
Alternaria cichorii		CBS 102.33; E.G.S. 07.017	Cichorium intybus, leaf spot	Cyprus	KJ718141	J717991	KJ718489
Alternaria cirsinoxia		CBS 113261; E.G.S. 41.136	Cirsium arvense, stem lesion	Canada, askatchewan	KJ718143	KJ717993	KJ718491
Alternaria citrullicola sp. nov.	Alternaria cucumerina	CBS 103.32; VKM F-1881	Citrullus vulgaris, fruit	Cyprus	KJ718144	KJ717994	KJ718492
Alternaria conidiophora sp. nov.		CBS 137457	–	Netherlands	KJ718145	KJ717995	KJ718493
Alternaria crassa		CBS 110.38	Datura stramonium, leaf spot	Cyprus	KJ718147	KJ717997	KJ718495
Alternaria cucumerina	Alternaria loofahae	CBS 116114; E.G.S. 35.123	Luffa acutangula	USA, Hawaii	KJ718153	KJ718000	KJ718501
Alternaria cyamopsidis		CBS 364.67; E.G.S. 17.065;	Cyamopsis tetragonoloba, leaf spot	USA, Maryland	KJ718156	KJ718003	KJ718504
		CBS 117219; E.G.S. 13.120;	Cyamopsis tetragonoloba, leaf spot	USA, Georgia	KJ718157	KJ718004	KJ718505
Alternaria dauci		CBS 111.38	Daucus carota, seed	Italy	KJ718158	KJ718005	KJ718506
	Alternaria cichorii	CBS 101592	Daucus carota, seed	Netherlands	KJ718162	KJ718009	KJ718510
		CBS 117099; E.G.S. 47.131	Daucus carota, seed	USA, California	KJ718164	KJ718011	KJ718512
Alternaria deserticola sp. nov.	Alternaria acalyphicola	CBS 110799	desert soil	Namibia	KJ718249	KJ718077	KJ718595
Alternaria dichondrae		CBS 199.74; E.G.S. 38.007	Dichondra repens, leaf spot	Italy	KJ718166	JQ646357	KJ718514
		CBS 200.74; E.G.S. 38.008	Dichondra repens, leaf spot	Italy	KJ718167	KJ718012	KJ718515
Alternaria echinaceae		CBS 116117; E.G.S. 46.081	Echinacea sp., leaf lesion	New Zealand, isborne	KJ718170	KJ718015	KJ718518
		CBS 116118; E.G.S. 46.082	Echinacea sp., leaf lesion	New Zealand, isborne	KJ718171	KJ718016	KJ718519
Alternaria grandis		CBS 109158; E.G.S. 44.106	Solanum tuberosum, leaf spot	USA, Pennsylvania	KJ718239	JQ646341	EU130547
		CBS 116695; E.G.S. 44.108	Solanum tuberosum, leaf spot	USA, Pennsylvania	KJ718241	KJ718070	KJ718587
Alternaria euphorbiicola		CBS 198.86; E.G.S. 38.082	Euphorbia pulcherrima	USA, Florida	KJ718172	KJ718017	KJ718520
		CBS 119410; E.G.S. 41.029	Euphorbia pulcherrima	USA, Hawaii	KJ718173	KJ718018	KJ718521
Alternaria gypsophilae		CBS 107.41; E.G.S. 07.025;	Gypsophila elegans, seed	Netherlands	KC584199	KC584118	KC584660
Alternaria ipomoeae sp. nov.	Alternaria cucumerina	CBS 219.79	Ipomoea batatas, stem	Ethiopia	KJ718175	KJ718020	KJ718523
		PPRI 8988	Ipomoea batatas, stem	South Africa, Gauteng	KJ718176	KJ718021	KJ718524
Alternaria jesenskae		CBS 133855; CCM 8361	Fumana procumbens, seed	Slovakia	KJ718177	KJ718022	KJ718525
Alternaria limicola		CBS 483.90; E.G.S. 39.070	Citrus aurantiifolia, leaf spot	Mexico, Colima	KJ718178	JQ646329	KJ718526
		CBS 117360; E.G.S. 43.009	Citrus sp.	Mexico, Jalisco	KJ718179	KJ718023	KJ718527
Alternaria linariae	Alternaria tomatophila	CBS 109156; E.G.S. 42.156	Solanum lycopersicum, leaf spot	USA, Indiana	KJ718183	JQ646347	KJ718531
	Alternaria subcylindrica	CBS 109161; E.G.S. 45.113	Solanum lycopersicum var. cerasiforme, leaf spot	USA, Louisiana	KJ718184	JQ646345	KJ718532
	Alternaria cretica	CBS 109164; E.G.S. 46.188	Solanum lycopersicum, leaf spot	Greece, Crete	KJ718185	JQ646342	EU130545
	Alternaria cucumericola	CBS 116438; E.G.S. 41.057	Cucumis sativus, leaf spot	New Zealand	KJ718186	KJ718027	KJ718533
Alternaria macrospora		CBS 117228; E.G.S. 50.190	Gossypium barbadense	USA, Arizona	KC584204	KC584124	KC584668
Alternaria montanica		CBS 121343; E.G.S. 44.112	Cirsium arvense	USA, Montana	KJ718194	KJ718033	KJ718541
Alternaria multirostrata		CBS 712.68; ATCC 18515	Richardia scabra, floral bract	USA, Georgia	KJ718195	JQ646362	EU130546
		CBS 713.68; ATCC 18517;	Richardia scabra, floral bract	USA, Georgia	KJ718196	KJ718034	KJ718542
Alternaria neoipomoeae sp. nov.		PPRI 11845	Ipomoea batatas, stem	South Africa, Gauteng	KJ718198	KJ718036	KJ718544
		PPRI 11847	Ipomoea batatas	South Africa, Mpumal	KJ718199	KJ718037	KJ718545
Alternaria nitrimali		CBS 109163; E.G.S. 46.151	Solanum viarum, leaf spot	Puerto Rico	KJ718201	JQ646358	KJ718547
Alternaria novae-guineensis		CBS 116120; E.G.S. 47.198	Citrus sp., dry leaf	Papua New Guinea	KJ718202	KJ718039	KJ718548
		PPRI 12171	Galinsoga parviflora, leaf	South Africa, Gauteng	KJ718203	KJ718040	KJ718549
Alternaria obtecta		CBS 117367; E.G.S. 42.063	Euphorbia pulcherrima, leaf	USA, California	KJ718204	KJ718041	KJ718550
		CBS 134278; E.G.S. 42.064	Euphorbia pulcherrima	USA, California	KJ718205	KJ718042	KJ718551
Alternaria paralinicola sp. nov.	Alternaria linicola	CBS 116652; E.G.S. 47.157;	Linum usitatissimum, seed	Canada, Manitoba	KJ718206	KJ718043	KJ718552
Alternaria passiflorae	Alternaria hawaiiensis	CBS 630.93; E.G.S. 29.020;	Passiflora edulis	USA, Hawaii	KJ718210	JQ646352	KJ718556
	Alternaria gaurae	CBS 116333; E.G.S. 50.121	Gaura lindheimeri, leaf	New Zealand, uckland	KJ718211	KJ718046	KJ718557
Alternaria pipionipisi		CBS 116115; E.G.S. 40.096	Cajanus cajan, seed	India	KJ718214	KJ718049	KJ718560
Alternaria porri	Alternaria allii	CBS 116649; E.G.S. 17.082	Allium cepa, leaf	USA, Nebraska	KJ718217	KJ718052	KJ718563
		CBS 116699; E.G.S. 48.152	Allium cepa, leaf spot	USA, New York	KJ718218	KJ718053	KJ718564
Alternaria protenta	Alternaria hordeiseminis	CBS 116437; E.G.S. 32.076	Hordeum vulgare, seed	New Zealand	KJ718220	KJ718055	KJ718566
	Alternaria solani	CBS 116651; E.G.S. 45.020	Solanum tuberosum, tuber	USA, California	KC584217	KC584139	KC584688
Alternaria pseudorostrata		CBS 119411; E.G.S. 42.060	Euphorbia pulcherrima	USA, California	JN383483	AY562406	KC584680
Alternaria ranunculi		CBS 116330; E.G.S. 38.039	Ranunculus asiaticus, seed	Israel	KJ718225	KJ718058	KJ718571
Alternaria ricini		CBS 215.31	Ricinus communis	Japan	KJ718226	KJ718059	KJ718572
		CBS 353.86	Ricinus communis	Italy, Sardinia	KJ718227	JQ646331	KJ718573
Alternaria rostellata		CBS 117366; E.G.S. 42.061	Euphorbia pulcherrima, leaf	USA, California	KJ718229	JQ646332	KJ718575
Alternaria scorzonerae	Alternaria linicola	CBS 103.46; Elliot No. 45-190C	Linum usitatissimum	UK, Scotland	KJ718190	JQ646363	KJ718537
		CBS 478.83; E.G.S. 38.011	Scorzonera hispanica, leaf spot	Netherlands, Reusel	KJ718191	JQ646334	KJ718538
	Alternaria linicola	CBS 116703; E.G.S. 36.110	Linum usitatissimum, seed	UK, Derbyshire	KJ718192	KJ718031	KJ718539
Alternaria sennae sp. nov.	Alternaria cassiae	CBS 477.81; E.G.S. 34.030	Senna corymbosa, leaf	India, Uttar Pradesh	KJ718230	JQ646344	EU130543
Alternaria sesami		CBS 240.73	Sesamum indicum	Egypt	KJ718231	JQ646343	KJ718576
		CBS 115264;	Sesamum indicum, seedling	India	JF780939	KJ718061	KJ718577
Alternaria sidae		CBS 117730; E.G.S. 12.129	Sida fallax, leaf spot	Kiribati, Phoenix islands	KJ718232	KJ718062	KJ718578
Alternaria silybi		CBS 134092; VKM F-4109	Silybum marianum, leaf	Russia, Vladivistok	KJ718233	KJ718063	KJ718579
		CBS 134093; VKM F-4117	Silybum marianum, leaf	Russia, Vladivistok	KJ718234	KJ718064	KJ718580
Alternaria solani	Alternaria danida	CBS 111.44; E.G.S. 07.029	Ageratum houstonianum, seed	Italy	Y17070	KJ718068	KJ718584
	Alternaria viciae-fabae	CBS 116442; E.G.S. 46.162	Vicia faba	New Zealand	KJ718240	KJ718069	KJ718586
Alternaria solani-nigri	Alternaria cyphomandrae	CBS 109155; E.G.S. 40.058	Cyphomandra betacea, fruit	New Zealand, New Plymouth	KJ718242	JQ646360	KJ718588
	Alternaria herbiculinae	CBS 116332; E.G.S. 49.180	Petroselinum crispum, plant	New Zealand, Taranaki	KJ718244	KJ718072	KJ718590
	Alternaria glyceriae	CBS 116334; E.G.S. 51.107	Glyceria maxima, leaf spot	New Zealand, Waikato	KJ718245	KJ718073	KJ718591
Alternaria steviae		CBS 631.88; IFO 31212	Stevia rebaudiana, leaf spot	Japan, Kagawa	KJ718250	KJ718078	KJ718596
		CBS 117362; E.G.S. 37.019	Stevia rebaudiana, leaf spot	Japan, Kagawa	KJ718252	KJ718079	KJ718598
Alternaria tagetica		CBS 297.79; GST AM2	Tagetes sp., seed	UK	KJ718253	KJ718080	KJ718599
		CBS 479.81; E.G.S. 33.081	Tagetes erecta, seed	UK, England	KC584221	KC584143	KC584692
		CBS 117217; E.G.S. 44.045	Tagetes sp., leaf spot	USA, Ohio	KJ718256	KJ718083	KJ718602
Alternaria thunbergiae		CBS 116331; E.G.S. 41.073	Thunbergia alata, leaf spot	Australia, Queensland	KJ718257	KJ718084	KJ718603
	Alternaria iranica	CBS 120986; E.G.S. 51.075	Allium cepa, leaf	Iran, Miandoab	KJ718258	KJ718085	KJ718604
		CBS 122597	Thunbergia alata	New Zealand, Auckland	KJ718259	KJ718086	KJ718605
Alternaria tillandsiae		CBS 116116; E.G.S. 43.074	Tillandsia usneoides	New Zealand	KJ718260	KJ718087	KJ718606
Alternaria tropica		CBS 631.93; E.G.S. 39.126	Passiflora edulis, fruit	USA, Florida	KJ718261	KJ718088	KJ718607
		CBS 117216; E.G.S. 39.125	Passiflora edulis, fruit	USA, Florida	KJ718262	KJ718089	KJ718608
Alternaria venezuelensis		CBS 116121; E.G.S. 48.065	Phaseolus vulgaris, leaf spot	Venezuela, Maracay	KJ718263	KJ718090	KJ718609
Alternaria zinniae		CBS 117223; E.G.S. 44.035	Zinnia elegans, leaf spot	New Zealand, Auckland	KJ718270	KJ718096	KJ718616

^aATCC: American Type Culture Collection, Manassas, VA, USA; BRIP: Queensland Plant Pathology Herbarium, Queensland, Australia; CBS: Culture collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Utrecht, Netherlands; CCM: Czech Collection of Microorganisms, Brno, Czech Republic; CECT: Spanish Type Culture Collection, Valencia, Spain; CPC: Personal collection of P.W. Crous, Utrecht, Netherlands; DAOM: Canadian Collection of Fungal Cultures, Ottawa, Canada; DSM: German Collection of Microorganisms and Cell Cultures, Leibniz Institute, Braunschweig, Germany; E.G.S.: Personal collection of Dr. E.G. Simmons; Elliott: Personal collection of M.E. Elliott; GST: Personal collection of G.S. Taylor; ICMP: International Collection of Micro-organisms from Plants, Auckland, New Zealand; IFO: Institute for Fermentation Culture Collection, Osaka, Japan; IMI: Culture collection of CABI Europe UK Centre, Egham UK; LEV: Plant Health and Diagnostic Station, Levin, New Zealand; MUCL: (Agro)Industrial Fungi and Yeast Collection of the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Louvain-la Neuve, Belgium; Nattrass: Personal collection of R.M. Nattrass; PD: Plant Protection Service, Wageningen, Netherlands; PPRI: ARC-Plant Protection Research Institute, Roodeplaat, South Africa; QM: Quarter Master Culture Collection, Amherst, MA, USA; VKM: All-Russian Collection of Microorganisms, Moscow, Russia.

2.5. Phylogenetic analyses

The obtained sequences and relevant ones available in the GenBank database (Table 1) were aligned using CLUSTAL_X ver. 2.0.11 (EMBL-EBI, Cambridgeshire, UK), and adjusted manually [21]. The combined dataset of ITS, GAPDH, and TEF1 gene sequences was analyzed using MEGA 6 software [22]. Maximum likelihood analysis was performed with 1000 bootstrap replicates and the General Time Reversible model of nucleotide substitution. The node reliability was assessed by no less than 70% of parsimony bootstrap support values A. gypsophilae (CBS107.41) were used as outgroups for the phylogeny analysis.

3. Results and discussion

3.1. Phylogenetic analysis

The combined dataset resulted in an alignment containing a total of 1434 characters, which comprised 583, 575, and 276 characters of ITS rDNA, GAPDH, and TEF1, respectively. Maximum likelihood analysis was used to generate the phylogenetic tree shown in Figure 1. The two isolates YITC 5109 and YITC 5114 of A. yunnanensis sp. nov. were identical in all these three genes, and formed a strongly supported clade in the phylogram, being most closely related to A. sidae, A sennae, A. deserticola. These two species further clustered together with A. cyamopsidis, A. rostellata, A. nitrimali, A. thunbergiae, and A. crassa.

Figure 1.

The Maximum-likelihood tree derived from combined ITS rDNA, GAPDH, and TEF1 gene sequences of Alternaria species. Bootstrap support values above 50% are shown at the nodes. Alternaria gypsophilae serve as outgroup. The strains YITC 5109 and YITC 5114 of a new species A. yunnanensis proposed in this study are emphasized in red.

3.2. Taxonomy

Alternaria yunnanensis Z.Y. Cai, X.Y. Liu, Y.X. Liu & Y.P. Shi, sp. nov. (Figure 2).

Figure 2.

Alternaria yunnanensis (ACCC 39327: ex-holotype). (A) Obverse (left) and reverse (right) of colony on PDA at 25 °C for 7 days; (B) Obverse (left) and reverse (right) of colony on V-8 juice agar at 25 °C for 7 days; (C) features of the sporulation apparatus; (D) Symptoms on Hevea brasiliensis leaves; (E) Conidia produced on V-8 juice agar at 22 °C for 7 days; (F) Conidiophores produced on V-8 juice agar at 22 °C for 7 days. Scale bars: 50 μm for (C), 40 μm for (E), 10 μm for (F).

Etymology: In reference to the location of Yunnan Province from where the fungus was isolated.

Fungal Name: FN570554.

Colonies of A yunnanensis on PDA agar develope well, attaining a diameter of 7.7–8.5 cm under a daily fluorescent light/dark cycle of 8/16 h at 25 °C in 7 days, circular, with evident concentric rings and a whitish aerial-mycelium margin, cottony, yellowish to light gray, secreting an orangish pigment into the medium (Figure 2(A)). Mycelia produced on V-8 agar are light to dark gray (Figure 2(B)). Conidiophores are simple or sparingly branched, septate, pale brown to mid brown, smooth, arising beneath the agar surface or directly from hyphae, ca. 40–104 × 5–7μm, and gradually enlarge near the apex into a clavate conidiogenous cell that produces conidia (Figure 2(F)). Conidia are abundant on V-8 agar, and few on PDA and PCA, smooth, dilute dull brown, 41–97 × 13–29 μm, with 5–10 transverse septa and 1–6 longitudinal or oblique septa, often constricted at the septa, usually solitary or rarely in chains of two (Figure 2(C,E)), straight or slightly curved, long ellipsoid to obclavate, tapering to the beak which is equal in length to or up to fourfold as long as the body. The apical beaks are simple, pale, septate, up to 252 μm long and 2.5 μm wide (Figure 2(E)).

3.3. Holotype

China, isolated from rubber tree leaf, Hula village, Ruili city, Yunnan Province, China, May 2014, Z.Y. Cai (dried culture HMAS 247784), ex-holotype cultures (ACCC 39327 and CGMCC3.18901).

3.4. Pathogenicity assay

Four days after inoculation, typical black leaf spots observed, which resembles the field symptom with round, black, foliar spots, surrounded by yellow halos, and lesions sometimes slightly sink (Figure 2(D)). A fungus reisolated from the leaf lesions on inoculated plants has the same colonial and conidial morphology as the inocula A. yunnanensis, but not from asymptomatic leaves of control rubber plants, fulfilling Koch’s postulates. The result indicates that the species is the causal agent of leaf spot diseases in rubber tree.

Based on phylogenetic analyses and morphological characteristics, the fungus isolated from the leaf spots of the rubber tree was identified. Both data indicate that the fungus is a novel species belonging to the genus Alternaria. With its long history of rubber cultivation, rubber trees have had ample opportunity to attract a wide variety of diseases. A number of significant new diseases have been discovered. At present, the Alternaria leaf disease is not very serious. However, identifying Alternaria species on rubber tree is important to control rubber tree disease in case the disease outbreaks. In China among the three known Alternaria disease of rubber tree, A. alternata is the most widely distributed, which can be found at every rubber planting region. Alternaria heveae and A. yunnanensis were only found at very few rubber plantations.

Along with the A. yunnanensis, 112 Alternaria strains including 63 species of the section Porri of Alternaria were analyzed. The phylogram (Figure 1) generated from the three-gene combined dataset (ITS, GAPDH, and TEF1) exhibits similar topology to previously reported one [3]. The phylogenetic analyses indicate that the new species, A. yunnanensis, belongs to the Porri species-group, all of which produce relatively large conidia with a simple or branched, filamentous long beak [2,3].

The Porri species-group includes 63 species [3]. Phylogenetic analysis showed that A. yunnanensis was most closely related to A. sidae, A sennae, A. deserticola, and relevant to A. cyamopsidis, A. rostellata, A. thunbergiae, A. nitrimali, and A. crassa in the section Porri.

Based on conidial morphology, A. yunnanensis is most similar to A. sennae. Conidia of A. yunnanensis have 5–10 transverse septa and often evidently constricted at the septa, whereas those of A. sennae have 7–8 transverse septa, slightly constricted near some transverse septa. Besides the conidia of A. sennae (55–62 × 10–12 μm) are narrower than that of A. yunnanensis (41–97 × 13–29 μm) [3]. The culture of A. deserticola (CBS 110799) is sterile. However, Conidia of A. yunnanensis are abundant on V-8 agar. Forthmore A. yunnanensis differs from A. deserticola of its closest phylogenetic neighbour based on alleles of all three loci (positions derived from respective alignments of the separate loci: ITS positions 389(C), 390(C), and 504(T); GAPDH positions 111(G), 172(C), 332(C), 482(T), and 500(C); TEF1 positions 264(C) and 310(A) [3]. Conidium outer walls of A. yunnanensis are smooth, while those of relevant species (A. sidae, A. cyamopsidis, A. rostellata, A. thunbergiae and A. nitrimali) are punctulate to strongly punctuate ornamentation. The conidia of A. yunnanensis are obviously smaller than that of A. thunbergiae (96–112 × 27–32 μm). Additionally, the apical beaks of A. yunnanensis are simple and long filamentous up to fourfold as long as the body, which differs from A. rostellata with a narrow beak, ca 30–55 × 2 µm or usually shorter, A. sennae with a single beak which occasinally branches once. A detailed comparison between A. yunnanensis and members of the Porri species-group is provided in Table 2.

Table 2.

Morphological characteristics of the present isolates and relevant species.

Species/MB or FN No.	Conidia				Beak
	Shape	Size (μm)	Septation	Ornamentation
A. yunnanensis FN 570554	Straight or curved, long ellipsoid to obclavate	41–97 × 13–29	5–10 transverse, 1–6 longitudinal or oblique septa	Smooth	A filamentous beak, up to 252 μm long and 2.5 μm wide
A. sidae MB 489129	Variant of a long-elliptical or sometimes cylindric	70–110 × 18–27	7–10 transverse septa and 1–2 longisepta	Conidia with a pustulate surface	A filamentous beak reach 95–110 × 1.5 µm
A. sennae MB 809000	Narrowly ovoid	55–62 × 10–12	7–8 transverse distosepta and 2–3 longitudinal septa	Smooth	A filamentous beak, occasionally branches once, ca.99–163 × 2 μm
A. cyamopsidis MB 292412	Broadly ovoid, ellipsoid, or sometimes obovoid	60–90 × 17–25	7–10 transverse distosepta with 1–3 longisepta	Conidia bear a scattering of granules	A filamentous beak up to 100–170 × 1.5–2.0 µm
A. rostellata MB 414600	Ellipsoid to broadly ovoid	50–80 × 20–30	7–9 transverse septa and 1–3 longitudinal septa	Smooth to moderately punctate-rough	A narrow beak, ca 30–55 × 2 µm or usually shorter
A. thunbergiae MB 504986	Broadly ovoid, ellipsoid, or obclavate	96–112 × 27–32	8–12 transverse septa and 1–2 longitudinal septa	Either smooth or, closely punctulate	tapered beak up to 225–320 × 2 µm
A. nitrimali MB 467495	Ovoid or ellipsoid,	70–87 × 14–17.5	7–10 transverse distosepta and only 1 longiseptum	Smooth or inconspicuously punctulate	A long filamentous beak 115–186 × 2 µm

Compared with other Alternaria pathogens on rubber tree, including A. alternata and A. heveae, the conidia of A. yunnanensis are quite different, which are obviously larger than that of A. alternata (22.5–67.5 × 10–15 µm) and A. heveae (22.75–49.5 × 8–20 µm) [12,13]. In GenBank, there are only ITS sequences of A. heveae and A. alternata (KF953884 and KM111289) of these known Alternaria pathogens of rubber tree. We compared our sequences with them, and they were different (data not shown). Thus, we confirm the novelty of A. yunnanensis.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This study was supported by the Founds for Technical Innovation Talents Training of Yunnan [2016HB016] and the Sci-Tech Innovation System Construction for Tropical Crops Grant of Yunnan Province [No. RF2017-6].