Z. Y. Cai's research while affiliated with Yunnan Nationality Institute and other places

Rubber tree (Hevea brasiliensis Muell. Arg.) is an economically important perennial in tropical areas of China, e.g., in Yunnan and Hainan provinces. During the summer of 2016, leaf spots were observed on rubber trees on two plantations located in Jingping and Jiangcheng counties in Yunnan Province. The surface area of the two plantations are approximately 6 ha, and around 8% of the trees were affected by the disease. Affected leaves showed black spots 1 to 3 mm in diameter surrounded by a yellow halo. Lesion tissues collected from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on potato dextrose agar (PDA), and incubated at 25°C in dark for 5 days. A fungus species was isolated from 81% of the infected tissues tested. Colonies of this fungus initially showed a white aerial mycelium, turning gray after 5 days, and dark gray after 7 days of incubation. The fungus produced black pycnidia with conidia after 7 days on double autoclaved leaves of rubber tree that were placed onto the surface of PDA. Conidia were hyaline, unicellular, ellipsoidal to fusiform, externally smooth, thin-walled, and ranged from 10.0 to 27.3 μm long (avg. 17.9 μm) × 2.6 to 9.7 μm wide (avg. 6.5 μm). Morphological characteristics of the isolates were similar to the descriptions of Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A. J. L. Philips reported in other studies (Crous et al. 2006; Phillips et al. 2013). It was identified as N. parvum. For further confirmation of the identity, the rDNA internal transcribed spacer (ITS) region, partial sequence of glyceraldeyde-3-phosphate dehydrogenase-like (GAPDH) gene, and beta-tubulin (TUB2) gene of a representative isolate JPZZP4 were amplified using the primer pairs ITS1/ITS4, GDF1/GDR1, and T1/Bt-2b, respectively. The sequences obtained were deposited in GenBank (accession nos. KY009942, KY009944, and KY009943 for ITS, GAPDH, and TUB2, respectively). Nucleotide BLAST analysis showed a 100% identity matching with N. parvum (KJ193662.1, KJ193646.1, KC816062.1, HQ332201.1) for ITS, 100% with N. parvum (GU251387.1) for GAPDH, and 100% with N. parvum (KU554657.1 and KU554656.1) for TUB2. Koch’s postulates were verified by inoculating five rubber trees (clone GT1) with the copper-colored leaf with a conidial suspension (10⁴conidia/ml) of the representative isolate JPZZP4. Five rubber plants sprayed with sterile distilled water served as noninoculated control plants. This inoculation experiment was repeated three times. All the plants were covered with plastic bags for 48 h to maintain high relative humidity and monitored daily for symptom development. Four days later, inoculated rubber plants showed black leaf spots and some defoliation. Control plants remained symptomless. The species N. parvum was reisolated from inoculated tissue, and had identical morphology compared with the initial isolate. To our knowledge, this is the first report of N. parvum causing disease on rubber tree worldwide. The diagnosis of this disease is obviously an essential step for eventually controlling this disease that poses a serious threat to the natural rubber industry. © 2017, American Phytopathological Society. All rights reserved.

In July 2015, a Colletotrichum species was isolated from rubber tree leaves (Hevea brasiliensis Muell. Arg.) growing at a rubber plantation in Dehong County, Yunnan Province, China. Green leaves of rubber tree showed anthracnose-like lesions surrounded by a ring of brown tissue. Necrotic leaf tissues turned gray and paper-like gradually. The disease incidence was 8%. Disks (0.5 cm) were cut around the necrotic sites and surface-sterilized with 75% ethanol and air-dried. Leaf disks were placed on potato dextrose agar (PDA) to establish cultures. Pure cultures were obtained by subculturing hyphal tips onto fresh PDA plates and grown under alternating near-UV light (12 h per day) and darkness at 25°C. The generated colonies had white aerial mycelia. The color of the reverse colony was reddish yellow. The average mycelial growth rate was 9.6 mm per day (n = 5). Microscopic observations identified the conidia as aseptate, cylindrical, hyaline, straight, truncated base, obtuse apex, 12.44 to 18.39 × 5.05 to 7.26 μm (n = 200). Morphological features suggested that these isolates possessed the same characteristics as previously described for Colletotrichum karstii (Damm et al. 2012; Yang et al. 2011). For molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA and partial sequence of β-tubulin genes of a representative isolate ML1a were amplified using the ITS1/ITS4 (White et al. 1990) and Bt2a/Bt2b (Glass and Donaldson 1995) primer pairs, respectively. Gene sequences were deposited in GenBank (accessions KU950746 for the ITS region; KU950747 for Bt2 region) and showed 99 to 100% identity to C. karstii isolate CBS 129833, CBS 129832, CBS 129829, and CBS129824 (JQ005175, JQ005177, JQ005189, and JQ005215 for ITS region and JQ005609, JQ005611, JQ005623, and JQ005649 for Bt2 region). Using the published ITS and tub2 data from C. karstii isolate (CBS 129824, CBS 129832, and CBS 129829), C. boninense isolate (CBS 129831, CBS 128547, and CBS 112115), C. gloeosporioides isolate (CBS 112999) (Damm et al. 2012), a phylogenetic tree was generated via the neighbor-joining method with bootstrap 500 replications. Evolutionary analyses were conducted in MEGA6. This created tree places the isolates in the same group as C. karstii. According to morphological and molecular characteristics, the pathogen was identified as C. karstii. Pathogenicity of a representative isolate, ML1a, was confirmed by inoculating three small rubber plants with copper-color leaf (∼5 to 7 cm in length). A spore suspension (∼1.6 × 10⁵ conidia/ml) was sprayed to the leaves using a manual pressure sprayer. Three small rubber plants sprayed with sterile distilled water were used as negative controls. After inoculation, the plants were covered with plastic bags. The plastic bags were removed 2 days postinoculation (dpi) and monitored daily for symptom development (Cai et al. 2013). Anthracnose-like symptoms were observed 6 dpi. The control plants remained healthy. A fungus with colony and conidial morphology similar to C. karstii were reisolated from leaf lesions on inoculated rubber plants, but not from asymptomatic leaves of control plants, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. karstii infection on rubber tree in china. © 2016, American Phytopathological Society. All rights reserved.

Natural rubber is one of the pillar industries of the tropical zone in China. In July 2015, leaf spot symptoms of rubber trees (Hevea brasiliensis Muell. Arg.) were observed at rubber plantations located in Danzhou, Hainan Province, and Maomin, Guangdong Province. Lesions began as tiny brown spots surrounded by a yellow halo. The lesions expanded gradually to circular or approximately circular necrosis with tawny to brown and gray center, 5 to 15 mm in diameter. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on potato dextrose agar (PDA) plates, and incubated at 25°C in alternating day/night cycles of natural light (∼11 h light, 13 h dark) for 4 days. Fungal colonies were caesious on PDA and produced abundant conidia on potato carrot agar plates. Conidiophores were single, cylindrical, geniculate, dark brown, and 5 to 7 µm thick with a swollen basal cell. Conidia were straight to slightly curved, ellipsoidal to narrowly obclavate orrostrate, brown, 6 to 16 distoseptate, with basal septum darker and thicker than other septum, 44.4 to 203.6 × 10 to 17.4 µm, with a distinctly protruding basal hilum. Germination of conidia were monopolar or bipolar. Based on morphological characteristics, the isolates were identified as Exserohilum rostratum (Drechs.) Leonard & Suggs (Ahmadpour et al. 2013; Michael et al. 1986). For molecular identification, genomic DNA of Er01DZS3 isolates from field samples were extracted from single conidial cultures and the internal transcribed spacer regions of rDNA (ITS1-5.8S-ITS4) were amplified and sequenced with primers ITS1 and ITS4 (White et al. 1990). BLASTn analysis revealed 100% sequence similarity with E. rostratum isolates (GenBank accession nos. KJ439663.1, GU966507.1, KP132777.1, KC150019.1, GU073109.1, and GQ478868.1). The ITS sequence of the Er01DZS3 isolate was deposited in GenBank (KU204880). Partial sequences of glyceraldeyde-3-phosphate dehydrogenase-like (Gpd) gene also were amplified and sequenced with primers GPD1 and GPD2. The partial sequence of the Gpd gene was deposited in GenBank (KU935741). BLASTn analysis revealed 99% sequence similarity with an E. rostratum isolate (AF081379.1). Morphologic characters and sequence analysis of the ITS rDNA and Gdp gene confirmed the pathogen was E. rostratum. Pathogenicity of the isolates was determined by fulfilling Koch’s postulates. Three rubber plants (the clone of rubber tree GT1) at the copper-colored leaf stage were stab inoculated with conidial suspension (10⁴ conidia/ml) of isolate Er01DZS3 and incubated at ∼30°C (avg.) with a natural photoperiod. Three plants were treated with sterile distilled water to serve as controls. After inoculation, the plants were covered with plastic bags to maintain high relative humidity. The plastic bags were removed 2 days after inoculation, and the plants were monitored daily for symptom development. All inoculated plants showed symptoms identical to those observed in the field 5 days after inoculation. No disease occurred on rubber plants used as the control. The fungus was reisolated from the diseased plants and confirmed to be E. rostratum based on morphological characteristics and sequence analysis. E. rostratum has been reported as causal agent of leaf spots on Zea mays, Ananas comosus, Calathea picturata, and Musa paradisiaca in China (Luo et al. 2012). To our knowledge, this is the first report E. rostratum causing disease on rubber tree in the China. This disease occurred sporadically on rubber plantations. The identification of pathogens is essential for disease management strategies. This report would establish a foundation for the further study of E. rostratum to address the disease effectively. © 2016, American Phytopathological Society. All rights reserved.

We first reported Alternaria heveae (E.G. Simmons ) to be the pathogen that caused black leaf spot of rubber tree (Hevea brasiliensis Muell. Arg) in Heikou county in July 2014 (1). Black leaf spots that resembled the symptoms caused by A. heveae were observed on the leaves of rubber trees of the whole propagule collection nursery in Jingping County (22°68′ N and 103°05′ E) of Yunnan Province. Black foliar spots (0.1 to 2 mm in diameter) surrounded by a yellow halo with lesions slightly sunken on the leaf surface were observed. To confirm whether the disease was caused by the same pathogen, 5-mm ² sections were removed from the leading edge of the lesion and were surface-sterilized in 75% ethanol, air-dried, plated on potato carrot agar (PCA), and incubated at 28°C in the dark. Colonies of the fungus on PCA had round margins and little aerial mycelia with gray-black coloration after 6 days of growth on PCA (2). Medium brown conidia were found to be in short chains of two to eight spores, ovoid, obclavate, and obpyriform, with or without a short conical or cylindrical-shaped apical beak. Conidia ranged from 22.5 to 67.5 μm long (mean 39.9 μm) × 10 to 15 μm wide (mean 12.5 μm; 100 colodia were measured), with three to six transverse septa and zero to three longitudinal or oblique septa. Morphological characteristics matched the descriptions of A. alternata [(Fries) Keissler] (4).The ITS1-5.8S-ITS2 region of one single-spore isolate, Ah02JP1, was amplified with primers ITS1 and ITS4. The PCR product was sequenced directly and deposited in GenBank (Accession No. KM111289). A BLAST search of the GenBank database revealed 100% similarity with A. alternata isolates KJ829535.1, KJ677246.1, and KF813070.1. Therefore, the pathogen was identified as A. alternata on the basis of its morphological characteristics and ITS sequence. Pathogenicity of a representative isolate, Ah02JP1 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage. Leaves were spray-inoculated (10 ⁴ conidia per milliliter spore suspension) until drops were equally distributed using a manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags to maintain high relative humidity. The plastic bags were removed 2 days post-inoculation (dpi), and the plants were monitored daily for symptom development. Five days post-inoculation, spots similar to the original ones seen on the field trees developed on all inoculated leaves, while control leaves remained symptomless. A. alternata was re-isolated from spray-inoculated leaves, confirming Koch's postulates. A. alternata has been reported as the causal agent of leaf blight of rubber tree in India, which initially appeared as minute spots on leaves and enlarged with the growth of the leaves (3). However, in the present study, the symptoms (black leaf spots) remained small over time after inoculation. To our knowledge, this is the first report of A. alternata on rubber tree in China. Correct identification of pathogens is essential for disease management strategies. This report will establish a foundation for the further study of Alternaria alternata to address the disease effectively. References: (1) Z. Y. Cai et al. Plant Dis. 98:1011, 2014. (2) E. Mirkova. J. Phytopathol. 151:323, 2003. (3) C. B. Roy et al. J. Plantation Crops 34:499, 2006. (4) T. Y. Zhang. Page 32 in: Flora Fungorum Sinicorum, Vol. 16: Alternaria. Science Press, Beijing, 2003.

Agrobacterium tumefaciens-mediated transformation (ATMT) is being increasingly recognized as an effective insertional mutagenesis tool in studies of filamentous fungi for gene discovery and functional analysis. We developed and optimized ATMT for 2 Colletotrichum species, Colletotrichum gloeosporioides and Colletotrichum acutatum, the causative agents of Colletotrichum leaf disease in rubber trees in Southern China. A. tumefaciens strain AGL-1 carrying an ILV1 gene and a green fluorescent protein gene were used to transform the conidia of these 2 Colletotrichum species. The transformation efficiency was correlated with the co-cultivation duration and bacterial cell concentrations, which reached 300–400 transformants per 1 × 106 conidia after optimization. Southern blot analysis indicated that about 60.0% of the C. gloeosporioides transformants and 46.2% of the C. acutatum transformants had a single copy of T-DNA in their genomes. Fungal genomic DNA segments flanking the T-DNA were identified in the transformants through thermal asymmetrical interlaced polymerase chain reaction followed by sequencing. The flanking sequences from 4 C. acutatum and 7 C. gloeosporioides transformants showed moderate or weak homology to the NCBI database entries. Some sequences matching those reported virulence-related genes. The results suggest that the T-DNA inserted mutants banks constructed are useful for the discovery of new or important genes and to elucidate their function in the 2 Colletotrichum species.