Morphological and Phylogenetic Analyses Reveal Three New Species of Didymella (Didymellaceae, Pleosporales) from Jiangxi, China.

Didymella contains numerous plant pathogenic and saprobic species associated with a wide range of hosts. Over the course of our mycological surveys of plant pathogens from terrestrial plants in Jiangxi Province, China, eight strains isolated from diseased leaves of four host genera represented three new species of Didymella, D. bischofiae sp. nov., D. clerodendri sp. nov., and D. pittospori sp. nov. Phylogenetic analyses of combined ITS, LSU, RPB2, and TUB2 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), revealed their taxonomic placement within Didymella. Both morphological examinations and molecular phylogenetic analyses supported D. bischofiae, D. clerodendri, and D. pittospori as three new taxa within Didymella. Illustrations and descriptions of these three taxa were provided, along with comparisons with closely related taxa in the genus.

Multi-locus phylogenetic analyses reveal eight novel species of Distoseptispora from southern China.

IMPORTANCE: Distoseptispora as a single genus in Distoseptisporaceae was introduced by morphological and phylogenetic analyses. Members of this genus occur mainly as asexual morphs, forming effuse, hairy colonies on decaying wood, plant stems, bamboo culms, and fallen leaves and shafts in terrestrial and freshwater habitats. In the present study, saprobic hyphomycetes from plant debris were investigated, and eight new Distoseptispora species were introduced based on morphology and phylogenetic analyses of LSU, ITS, TEF1, and RPB2 sequence data. This study provides important data on the species diversity, ecological environment, and geographical area of Distoseptispora, greatly updates the classification of Distoseptispora, and improves our understanding of the taxonomy of Distoseptispora.

First Report of Colletotrichum fructicola Causing Anthracnose on Punica granatum in China.

Punica granatum L. (Pomegranate), a deciduous shrub, is widely cultivated as a fruit tree and decorative plant in China. Its flowers, leaves, roots and fruit bark also has been widely used for the treatment of different types of human disease because of the high anti-inflammatory and antibacterial activitiy (Tehranifar et al. 2011). In October 2022, leaf spot symptoms were observed on P. granatum leaves in a landscaped area on the campus of Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. A survey of 40 P. granatum of 300 m2 found that up to 20% of the foliage was infected. Infection normally starts at the tip or edge of the leaves, with the initial symptoms of lesions usually being small dark brown spots (0.8 to 1.5 mm) that gradually expand into irregular spots with grayish white central parts, and brown margins (2.3 to 3.8 mm). Ten freshly infected leaves from three different plants were collected and cut into small slices, disinfected with 75% ethanol for 30 seconds followed by 5% NaClO for 1 minute, rinsed 3 times with sterile water, and then plated on potato dextrose agar (PDA) and incubated in the dark at 25°C. After 7 days, all incubated samples produced similar morphology of aerial mycelium pale grey, dense, and cottony. Conidia were hyaline, smooth-walled, cylindrical, aseptate and measuring 12.28 to 21.05 × 3.51 to 7.37 µm (n = 50). Morphological characteristics were consistent with those of Colletotrichum gloeosporioides species complex (Weir et al. 2012; Park et al. 2018). For molecular identification, we used two representative isolates (HJAUP CH005 and HJAUP CH006) for genomic DNA extraction and amplification, using primers for ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b, GDF1/GDR1, ACT-512F/ACT-783R and CL1C /CL2C (Weir et al. 2012), respectively. The sequenced loci (GenBank accession nos. ITS: OQ625876, OQ625882; TUB2: OQ628072, OQ628073; GAPDH: OQ628076, OQ657985; ACT: OQ628070, OQ628071; CAL: OQ628074, OQ628075) exhibited 98 to 100% homology with corresponding sequences of C. fructicola strains (GenBank accession nos. OQ254737, MK514471, MZ133607, MZ463637, ON457800, respectively). A phylogenetic tree was constructed using the maximum-likelihood method in MEGA7.0 for the sequences of five concatenated genes (ITS-TUB2-GAPDH-ACT-CAL). Our two isolates clustered together with three strains of C. fructicola with 99% bootstrap support values in the bootstrap test (1000 replicates). The isolates were identified as C. fructicola based on morpho-molecular approach. The pathogenicity of HJAUP CH005 was tested indoors by inoculating the wounded leaves of four healthy P. granatum plants. Four leaves from each of two healthy plants were punctured with flamed needles and sprayed with a spore suspension (1 × 106 spores/ml), and four wounded leaves from each of other two plants were inoculated with mycelial plugs (5 × 5 mm3), respectively. Mock inoculations with sterile water and PDA plugs on four leaves each were used as controls. Treated plants were incubated in a greenhouse at high relative humidity, 25°C, and a photoperiod of 12 h. After 4 days, typical anthracnose symptoms similar to natural infection appeared on the inoculated leaves, whereas the control leaves remained asymptomatic. Based on morphological and molecular data, the fungus isolated from the inoculated and symptomatic leaves was identical to the original pathogen, confirming Koch's hypothesis. Anthracnose caused by C. fructicola has been reported to affect numerous plants worldwide, including cotton, coffee, grapes and citrus (Huang et al. 2021; Farr and Rossman 2023). This is the first report of C. fructicola causing anthracnose on P. granatum in China. This disease seriously affects the quality and yield of the fruit and should be of wide concern to us.

First Report of Anthracnose Caused by Colletotrichum siamense on Hydrangea macrophylla in China.

Hydrangea macrophylla (Thunb.) Ser. (Hydrangeaceae), a shrubby perennial plant, is widely used as an ornamental flowering plant because of its showy inflorescences and colorful sepals. In October 2022, leaf spot symptom was observed on H. macrophylla in Meiling Scenic Spot, which covers an area of about 143.58 km2 in Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). An investigation was carried out in a 500 m2 mountain area with 60 H. macrophylla plants in a residential garden, the incidence of disease observed was 28~35%. The symptoms were visible as nearly round dark brown spots on the leaves in the early stages of infection. At later stages, the spots gradually developed grayish white center with dark brown margins. To isolate the pathogen, seven leaves randomly selected from 30 infected leaves were cut into 4-mm2 pieces, surface disinfected with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and cultured at 25 °C in the dark for 7 days, and four strains with similar morphological characteristics were obtained from 7 diseased samples. Conidia were aseptate, cylindrical, hyaline, obtuse at both ends, and measured 13.31 to 17.53 × 4.43 to 7.45 µm (15.47 ± 0.83 × 5.91 ± 0.62 µm, n = 60). Morphological characteristics matched Colletotrichum siamense (Weir et al. 2012; Sharma et al. 2013). For molecular identification, two representative isolates (HJAUP CH003 and HJAUP CH004) were used for genomic DNA extraction, and the internal transcribed spacer (ITS), partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2) and partial calmodulin (CAL) were amplified, using primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R, GDF1/GDR1, Bt2a/Bt2b and CL1C/CL2C (Weir et al. 2012), respectively. The sequences were deposited in GenBank (accessions nos. ITS: OQ449415, OQ449416; ACT: OQ455197, OQ455198; GAPDH: OQ455203, OQ455204; TUB2: OQ455199, OQ455200; CAL: OQ455201, OQ455202). Concatenated sequences of the five genes were used to conduct phylogenetic analyses using the maximum-likelihood method in MEGA7.0 (Sudhir et al. 2016) and Bayesian inference analysis in MrBayes 3.2 (Ronquist et al. 2012). Our two isolates cluster together with four strains of C. siamense with 93%ML/1.00BI bootstrap support. The isolates were identified as C. siamense based on the morpho-molecular approach. Pathogenicity of HJAUP CH003 was tested indoors by inoculating detached wounded leaves of six healthy H. macrophylla plants. Three healthy plants with three leaves were punctured with flamed needles and sprayed with a 1 × 106 spores/ml spores suspension, and another three healthy plants were wounded inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each. Treated plant tissue were incubated in an artificial climate box at 25°C, 90% relative humidity and a photoperiod of 12 h. After 4 days, symptoms similar to those of natural infection were observed on all wounded inoculated leaves, while no symptoms appeared on mock-inoculated leaves. The fungus isolated from inoculated leaves was identical to the original pathogen based on morphological and molecular data, confirming Koch's hypothesis. It has been reported that C. siamense can cause anthracnose on numerous plants (Rong et al. 2021; Tang et al. 2021; Farr and Rossman 2023). This is the first report of C. siamense causing anthracnose on H. macrophylla in China. The disease is of major concern to the horticultural community as it seriously affects the aesthetic value of ornamentals.

Morphological and Phylogenetic Analyses Reveal Three New Species of Distoseptispora (Distoseptisporaceae, Distoseptisporales) from Yunnan, China.

Three new species of Distoseptispora, viz. D. mengsongensis, D. nabanheensis, and D. sinensis, are described and illustrated from specimens collected on dead branches of unidentified plants in Yunnan Province, China. Phylogenetic analyses of LSU, ITS, and TEF1 sequence data, using maximum-likelihood (ML) and Bayesian inference (BI), reveal the taxonomic placement of D. mengsongensis, D. nabanheensis, and D. sinensis within Distoseptispora. Both morphological observations and molecular phylogenetic analyses supported D. mengsongensis, D. nabanheensis, and D. sinensis as three new taxa. To extend our knowledge of the diversity of Distoseptispora-like taxa, a list of recognized species of Distoseptispora with major morphological features, habitat, host, and locality is also provided.

First Report of Corynespora cassiicola Causing Leaf Spot on Jasminum nudiflorum in China.

Winter jasmine (Jasminum nudiflorum Lindl.), a trailing, deciduous shrub, is widely used as an ornamental plant. Its flowers and leaves also has great medicinal value for treatment of inflammatory swelling, purulent eruptions, bruises and traumatic bleeding (Takenaka et al. 2002). In October 2022, leaf spot symptoms were observed on J. nudiflorum distributed in Meiling Scenic Spot (28.78°N, 115.83°E) and Jiangxi Agricultural University (28.75°N, 115.83°E), Nanchang, Jiangxi Province, China. In a week-long series of investigations, the incidences of disease could range up to 25%. Initially, the symptoms of the lesions were small yellow circular spots (0.5 to 1.8 mm), and gradually developing irregular spots (2.8 to 4.0 mm) with grayish white central parts, a dark brown inner ring, and outer yellow halo. To identify the pathogen, sixty symptomatic leaves from fifteen different plants were collected, of which twelve were randomly selected, cut into 4-mm2 pieces, and surface sterilized with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed four times with sterile water, and then placed on potato dextrose agar (PDA) medium at 25 °C in the dark for 5 to 7 days. Six isolates with similar morphological characteristics were obtained. Aerial mycelium was vigorous, downy and exhibited white to grayish-green coloration. Conidia were solitary or catenate, pale brown, obclavate to cylindrical, apex obtuse, one to 11 pseudosepta, 24.9 to 125.7 × 7.9 to 12.9 µm (n = 50). Morphological characteristics matched Corynespora cassiicola (Ellis 1971). For molecular identification, two representative isolates (HJAUP C001 and HJAUP C002) were selected for genomic DNA extraction, and the ITS, TUB2 and TEF1-α gene were amplified, using the primer ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Lousie and Donaldson 1995) and EF1-728F/EF-986R (Carbone and Kohn 1999), respectively. The sequenced loci (GenBank accession nos. ITS: OP957070, OP957065; TUB2: OP981639, OP981640; TEF1-α: OP981637, OP981638) of the isolates were 100, 99 and 98% similar to the corresponding sequences of C. cassiicola strains (GenBank accession nos. OP593304, MW961419, MW961421, respectively). Phylogenetic analyses of combined ITS and TEF1-α sequences was performed using maximum-likelihood method in MEGA 7.0 (Kuma et al. 2016). The result showed that our isolates (HJAUP C001 and HJAUP C002) clustered with four strains of C. cassiicola at 99% bootstrap values in the bootstrap test (1000 replicates). Based on the morpho-molecular approach, the isolates were identified as C. cassiicola. The pathogenicity of one representative strain (HJAUP C001) was tested by inoculating the wounded leaves of six healthy J. nudiflorum plants under natural condition. Three leaves from each of three plants were punctured with flamed needles and sprayed with a conidial suspension (1 × 106 conidia/ml), and three wounded leaves from each of other three plants were inoculated with mycelial plugs (5 × 5 mm3). Mock inoculations were used as controls with sterile water and PDA plugs on three leaves each, respectively. Leaves from all treatments were incubated in a greenhouse at high relative humidity, 25°C, and 12-hour photoperiod. After one week, all wounded inoculated leaves appeared similar symptoms as described above, whereas the mock inoculated leaves were still healthy. Similar isolates with grayish white and vigorous aerial mycelium were reisolated from inoculated and symptomatic leaves and identified as C. cassiicola by DNA sequencing, fulfilling Koch's postulates. It has been reported that C. cassiicola can cause leaf spots on numerous plant species (Tsai et al. 2015; Lu et al. 2019; Farr and Crossman 2023). However, to our knowledge, this is the first report of C. cassiicola causing leaf spots on J. nudiflorum in China. This finding aids in protection of J. nudiflorum, a medicinal and ornamental plant with high economic value.

Morphology and Multigene Phylogeny Revealed Three New Species of Helminthosporium (Massarinaceae, Pleosporales) from China.

Saprobic hyphomycetes are highly diverse on plant debris. Over the course of our mycological surveys in southern China, three new Helminthosporium species, H. guanshanense sp. nov., H. jiulianshanense sp. nov. and H. meilingense sp. nov., collected on dead branches of unidentified plants, were introduced by morphological and molecular phylogenetic analyses. Multi-loci (ITS, LSU, SSU, RPB2 and TEF1) phylogenetic analyses were performed using maximum-likelihood and Bayesian inference to infer their taxonomic positions within Massarinaceae. Both molecular analyses and morphological data supported H. guanshanense, H. jiulianshanense and H. meilingense as three independent taxa within Helminthosporium. A list of accepted Helminthosporium species with major morphological features, host information, locality and sequence data was provided. This work expands our understanding of the diversity of Helminthosporium-like taxa in Jiangxi Province, China.

Molecular Phylogeny and Morphology Reveal Four Novel Species of Corynespora and Kirschsteiniothelia (Dothideomycetes, Ascomycota) from China: A Checklist for Corynespora Reported Worldwide.

Plant debris are habitats favoring survival and multiplication of various microbial species. During continuing mycological surveys of saprobic microfungi from plant debris in Yunnan Province, China, several Corynespora-like and Dendryphiopsis-like isolates were collected from dead branches of unidentified perennial dicotyledonous plants. Four barcodes, i.e., ITS, LSU, SSU and tef1-α, were amplified and sequenced. Morphological studies and multigene phylogenetic analyses by maximum likelihood and Bayesian inference revealed three new Corynespora species (C. mengsongensis sp. nov., C. nabanheensis sp. nov. and C. yunnanensis sp. nov.) and a new Kirschsteiniothelia species (K. nabanheensis sp. nov.) within Dothideomycetes, Ascomycota. A list of identified and accepted species of Corynespora with major morphological features, host information and locality was compiled. This work improves the knowledge of species diversity of Corynespora and Kirschsteiniothelia in Yunnan Province, China.

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To elucidate the composition of semiochemicals of Apriona germari and its function in sexual communication, GC-MS was used to detect the composition of semiochemicals of the overall body and the end abdominal tissue extracts in A. germari. Y-tube olfactometer was used to determine the olfactory response of adult female and male to the standard compounds of the five main extracts. The contact reaction test with male and female adults was performed to the eluted adults that smeared tandard compounds. The results showed that the main ingredients of semiochemicals were alkanes and alkenes with more than 10 carbons. Concentration of (Z)-9-Tricosene was the highest, followed by heptacosane, nonacosane, nonadecene, octacosane, 9-Hexylheptadecane, aldehyde, and ester. Results of the olfactory reaction showed that nonacosane had a significant attractivity to both male and female adults, and that heptacosane had a significant attractivity only to female adults. Nonadecene had a extremely significant repellent activity to female adults. 1-docosene and (Z)-9-Tricosene had no evident role to the male and female adults. Results of the contact test showed that male adults had the strongest courtship responses to the eluted adults with 1-docosene, heptacosane and nonacosane. Female adults had the strongest courtship responses to the eluted adults with nonacosane. Our results indicated that 1-docosene, heptacosane, and nonacosane were important component of the sex pheromone of A. germari, which played an important role in the sexual communication.

Variation in the transmission of barley yellow dwarf virus-PAV by different Sitobion avenae clones in China.

Fourteen Sitobion avenae Fabricius (Hemiptera: Aphididae) clonal lines (clones) originating from China were tested for their ability to transmit BYDV-PAV (one isolate from Belgium and another from China) using wheat plants. By sequence analysis, the coat protein gene of BYDV-PAV-BE was distinguishable from BYDV-PAV-CN. All of the clones could transmit BYDV-PAV, and the transmission varied from 24.42% to 66.67% with BYDV-PAV-BE and from 23.55% to 56.18% with BYDV-PAV-CN. These data suggest that S. avenae has no specialty in BYDV-PAV isolate. Significant differences in the transmission frequencies between the clones with BYDV-PAV-BE and BYDV-PAV-CN were observed. The transmission efficiencies of aphid clones from the middle-lower reaches of Yangtze River (AH, HD, HDE, HZ, JZ, JY and SJ) and Yunnan province (YH) were similar. Nevertheless, differences in the virus transmission efficiencies of the clones from northern (ST and STA) and northwestern (QX, SB and XS) regions were assessed. The transmission efficiency of S. avenae from northern and northwestern China, where BYDV impact is more important, was higher than that from the middle-lower reaches of the Yangtze River and Yunnan province. This work emphasizes the importance of considering aphid vector clonal diversity in addition to virus strain variability when assessing BYDV transmission efficiency.

Discovery of English grain aphid (Hemiptera: Aphididae) biotypes in China.

The English grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae), is an important pest insect of wheat, Triticum aestivum (L.), in China. Grain aphid biotypes are necessary to breed aphid-resistant wheat varieties; however, none have currently been identified. Here, we describe a method to identify grain aphid biotypes and survey the aphid biotype variation in the wheat growth area of China. Clones of S. avenae were collected from 11 locations in China and used to establish culture populations. These populations were then used to assess the resistance of 12 wheat varieties. Based on resistance responses, seven differential hosts were selected to identify the biotype of S. avenae: Amigo, 'Fengchan No. 3', Zhong 4 wumang, JP1, L1, 885479-2, and 'Xiaobaidongmai'. S. avenae was ultimately classified into five biotypes: EGA I, EGA II, EGA III, EGA IV, and EGA V. These methods provide a mechanism to detect the variation and evolution of grain aphids in different wheat-growing locations and also allow for selection of appropriate aphid-resistant germplasm for wheat breeding of commercial wheat cultivars.

Cellular tropism, population dynamics, host range and taxonomic status of an aphid secondary symbiont, SMLS (Sitobion miscanthi L type symbiont).

SMLS (Sitobion miscanthi L type symbiont) is a newly reported aphid secondary symbiont. Phylogenetic evidence from molecular markers indicates that SMLS belongs to the Rickettsiaceae and has a sibling relationship with Orientia tsutsugamushi. A comparative analysis of coxA nucleotide sequences further supports recognition of SMLS as a new genus in the Rickettsiaceae. In situ hybridization reveals that SMLS is housed in both sheath cells and secondary bacteriocytes and it is also detected in aphid hemolymph. The population dynamics of SMLS differ from those of Buchnera aphidicola and titer levels of SMLS increase in older aphids. A survey of 13 other aphids reveals that SMLS only occurs in wheat-associated species.

A possibly new Rickettsia-like genus symbiont is found in Chinese wheat pest aphid, Sitobion miscanthi (Hemiptera: Aphididae).

In this study, we investigated Rickettsia infection in Chinese wheat pest aphid (Sitobion miscanthi), moreover detected a possibly new Rickettsia-like symbiont, provisionally named as SMLS(1) (S. miscanthi L type symbiont). The sequence of SMLS 16S rRNA gene is 94% similar to that of its presumed closest relative, Orientia tsutsugamushi. If levels of divergence indicate taxonomic distinctiveness, SMLS probably represents a new genus in the family Rickettsiaceae. SMLS occurs in most populations of S. miscanthi, and with divergent infection frequencies, from 5.0% to 93.8%.