This study led to the discovery of three entomopathogenic fungi associated with Kuwanaspis howardi, a scale insect on Phyllostachys heteroclada (fishscale bamboo) and Pleioblastus amarus (bitter bamboo) in China. Two of these species belong to Podonectria: P. kuwanaspidis X.L. Xu & C.L. Yang sp. nov. and P. novae-zelandiae Dingley. The new species P. kuwanaspidis has wider and thicker setae, longer and wider asci, longer ascospores, and more septa as compared with similar Podonectria species. The morphs of extant species P. novae-zelandiae is confirmed based on sexual and asexual morphologies. Maximum likelihood and Bayesian inference analyses of ITS, LSU, SSU, tef1-α, and rpb2 sequence data provide further evidence for the validity of the two species and their placement in Podonectriaceae (Pleosporales). The second new species, Microcera kuwanaspidis X.L. Xu & C.L. Yang sp. nov., is established based on DNA sequence data from ITS, LSU, SSU, tef1-α, rpb1, rpb2, acl1, act, cmdA, and his3 gene regions, and it is characterized by morphological differences in septum numbers and single conidial mass.
In the present study, we surveyed the ascomycetes from bamboo of Phyllostachys across Sichuan Province, China. A biphasic approach based on morphological characteristics and multigene phylogeny confirmed seven species, including one new genus, two new species, and five new host record species. A novel genus Paralloneottiosporina is introduced to accommodate Pa. sichuanensis that was collected from leaves of Phyllostachys violascens. Moreover, the newly introduced species Bifusisporella sichuanensis was isolated from leaves of P. edulis, and five species were newly recorded on bamboos, four species belonging to Apiospora, viz. Ap. Yunnana, Ap. Neosubglobosa, Ap. Jiangxiensis, and Ap. Hydei, and the last species, Seriascoma yunnanense, isolated from dead culms of P. heterocycla. Morphologically similar and phylogenetically related taxa were compared. Comprehensive descriptions, color photo plates of micromorphology are provided.
Juglans sigillata Dode (Iron walnut) is mostly distributed in southwestern China, and valued for wood and nuts (Feng et al. 2018). In April 2020, we surveyed a walnut garden located in Baisha Town, Wanyuan City, (Sichuan, China), where brown spot symptoms were observed on leaves of ten trees among of 100 plants, and this disease can result in a reduced growth potential when trees are severely infected. Necrotic and subcircular lesions with conidiamata were observed on diseased leaves. Symptomatic leaves were collected and taken back to the laboratory forfurther analysis. Using the single spore isolation technique developed by Chomnunti et al. (2014), five isolates were grown from the infected leaves on Potato Dextrose Agar medium (PDA). The five isolates had similar colony morphology, which was initially white, suborbicular, gradually turning yellowish with black spots, developing fluffy aerial mycelium. Morphological characteristics were examined using light microscopy on the PDA. Conidiogenous cells were subcylindrical to cylindrical, or ampulliform, hyaline, rarely branched. Macroconidia were lunate, reniform, hyaline, 1-3-septate, mostly 1-septate, distinctly constricted at the septum, the basal cell was bluntly rounded, the apical cell had an acute end, and the basal cell was equal to or larger than the apical cell, measuring 22 to 40.5 × 2.5 to 8.3 μm (mean = 32 × 6.2 μm, n = 50). Microconidia were botuliform, or subfusiform, hyaline, both ends were rounded, straight or curved, aseptate, and measured 10 to 28.5 × 1.9 to 3.7 μm (mean= 17.2 × 2.7 μm, n = 20). A multilocus approach was conducted for precise identification of a representative isolate SICAUCC 20-0012. The internal transcribed spacer regions (ITS), guanine nucleotide-binding protein subunit beta gene (MS204), and translation elongation factor 1-alpha (tef1-α) of isolate SICAUCC 20-0012 were amplified and sequenced as described by Sogonov et al. (2008) and Walker et al. (2012a). GenBank Accession Nos. for ITS, MS204, and tef1-α are MW250303, MW246773, and MW246775, respectively. Phylogenetic analyses showed 100% support with Ophiognomonia leptostyla (Fr.) Sogonov, and the morphology was consistent with the asexual stage of O. leptostyla documented by Walker et al. (2012b). To test Koch’s postulates, five healthy plants of J. sigillata (2- to 3-year-old) with 5-8 leaves per plant were inoculated with conidial suspensions (104 conidia/mL) after wounded with a small pin as described by Desai et al. (2019), and the same number of healthy plants were wounded and sprayed with sterile distilled water as controls. Plants were sprayed regularly with distilled water every day and placed in a growth chamber at 25℃ with a 12-h fluorescent light/dark regimen. After 15 days, typical brown spot symptoms developed on inoculated leaves, but not on the controls. The fungus O. leptostyla was reisolated from the lesion as described above but not from non-inoculated leaves. O. leptostyla has been reported on some walnut trees; for example: J. ailantifolia, J. californica, J. cinerea, J. hindsii, J. major, J. mandshurica, J. nigra, and J. regia (Farr & Rossman 2020). However, to our knowledge, this is the first report of O. leptostyla causing brown leaf spot on J. sigillata. J. sigillata is an economically important tree in southwest China, and fungicide treatments should be considered to prevent the spread of this fungus before it becomes more widespread. Chunlin Yang, Yu Deng, and Feihu Wang contributed equally to this work. This research was supported by the Key Research and Development Project of Sichuan Province (2021YFYZ0032)
“Chuanzao 2” is a walnut variety derived from the hybridization of Juglans regia L. and J. sigillata Dode distributed in southwest China, where it is an economically important tree species in rural regions (Xiao et al. 2012). In April 2020, the variety in a walnut garden showed symptoms of brown leaf spot in Beishan Town (107°21′43.93″E, 31°28′12.34″N), Dazhou City in Sichuan, China, with 5% to 10% of leaves per plant affected (5 plants). Symptomatic leaves showed brown to dark brown spots (2 to 5 mm) with a dark brown to black halo and grayish-tan center. The spots were subcircular to irregular in shape, and gradually expanded and formed necrotic spots. A single conidium isolation was performed (Senanayake et al. 2020) and transferred to Potato Dextrose Agar (PDA). Five isolates were obtained from five different infected leaves. Colonies of five isolates were subcircular, erose or dentate, flat or effuse, white initially, gradually becoming yellowish with white margins, developed and fluffy aerial mycelia, and conidiogenensis was produced underneath mycelia after 25-days-incubation. Conidiogenous cells were subcylindrical to cylindrical, or irregular in shape, and hyaline. Macroconidia were lunate, reniform, hyaline, basal cell bluntly rounded, apical cell with acute end, 1-septate, rarely aseptate, sometimes slightly constricted at septum, basal cell equal or larger than apical cell, and measured 16.5 to 30.5 × 5 to 8.5 μm (mean = 23.2 × 6.3 μm, n = 50). Microconidia were not observed. These morphological characteristics resembled those of Ophiognomonia leptostyla (Fr.) Sogonov (Walker et al. 2012a). For molecular identification, genomic DNA (isolates SICAUCC 21-0008 and SICAUCC 21-0010) was extracted, and the internal transcribed spacers (ITS) region, guanine nucleotide-binding protein subunit beta (MS204) gene, and translation elongation factor 1-alpha (tef1-α) were amplified and sequenced by using the primers ITS5/ITS4 (White et al. 1990), E1F1/E5R1a (Walker et al. 2012a), and EF1-728F/EF1-1567R (Walker et al. 2012b), respectively. Phylogenetic analyses (maximum likelihood) based on a combined dataset showed 100% bootstrap support values in a clade with O. leptostyla. The sequences of ITS, MS204, and tef1-α genes were deposited in GenBank with accession numbers MW493111/MZ026300, MW495270/MZ031975, and MW495271/MZ031974, respectively. To fulfill Koch’s postulates, five healthy hybrid plants (2 to 3 years old) with 5 to 8 leaves per plant were spray inoculated with conidium suspensions (104 conidia/mL; isolate SICAUCC 21-0008) prepared from 40-days-old cultures onto the wounded sites via pin-prick inoculation. Similarly, five noninoculated plants sprayed with sterile water served as controls. Plants were placed in a growth chamber at 25℃ on a 12-h fluorescent light/dark regime and daily sprayed with sterile distilled water. After two weeks, observed symptoms were similar to those from natural infections. No disease symptoms were found on control plants. The fungus O. leptostyla was reisolated from the diseased leaves and characterized morphologically. O. leptostyla is a global pathogen and has been reported to cause the leaf spot in many walnut trees, viz. J. ailantifolia, J. californica, J. cinerea, and J. major, etc. To our knowledge, this is the first report of O. leptostyla causing brown leaf spot on Juglans hybrid (J. regia × J. sigillata) in China. The increasing risk of this pathogen in the walnut-growing areas of Sichuan Province of China needs a further exploration and outreach effort to develop effective control measures. Chunlin Yang, Feng Liu, and Qian Zeng contributed equally to this paper.
Phyllostachys aureosulcata McClure 'Spectabilis' C.D. Chu. et C.S. Chao is predominantly native to subtropical to warm temperate areas and is widely cultivated for landscaping in China (Neményi et al. 2015). In November 2020 (10 – 16 ℃), culm blight symptoms were observed on P. aureosulcata 'Spectabilis' in Wangjiang Tower Park (all kinds of plant areas are about 9.8 ha), Chengdu City (104°09′30.42″ E, 30°63′18.89″ N). Fifty plants were surveyed, and disease incidence was recorded as approximately 30%. Initially, chlorotic necrotic patches appeared on the culms, and gradually the patches became white, expanded to both ends, and encircled the whole culm with black edge and conidiomata, which eventually led to wilt and death. Five samples from different bamboos were collected and one of them were used for morphological observation. Five single conidia isolates were carried out on potato dextrose agar (PDA) at 25±1℃ (Chomnunti et al. 2014). Colonies were initially white and then yellowish in the center with abundant aerial mycelia. On the culm, conidiomata were dry, black, and filamentous. Conidiophores were reduced to conidiogenous cells. Conidiogenous cells were smooth, hyaline, ampulliform to doliiform. Conidia were ellipsoid to globose, dark brown, smooth and aseptate, measuring 5.2 to 9.4 × 4.4 to 7.3 μm, (=8.2 × 6.5μm, n=50). On the PDA medium, conidia were globose to subglobose, olive green to pale brown, and smooth, larger than those from the host in size, measuring 9.0 to 18 × 7.5 to 9.5 μm ( =36.6 × 18.8 μm, n=50). These asexual structures were extremely similar to Apiospora locuta-pollinis (F. Liu & L. Cai) X.G. Tian & Tibpromma (Zhao et al. 2018). DNA was extracted from the representative strain (SICAUCC 22-0036), and the internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α), beta-tubulin (tub2), 28S large subunit rDNA (LSU) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F (Carbone & Kohn 1999)/EF2 (O’Donnell et al. 1998), T1 (O’Donnell & Cigelnik 1997)/Bt2b (Glass & Donaldson 1995) and LR0R/LR5 (Rehner & Samuels 1994). The newly generated sequences were deposited in GenBank with accession nos. ON228609 (ITS), ON324018 (tef1-α), ON237657 (tub2), and ON228665 (LSU). Nucleotide blast showed 98.97%, 100% and 99.46% identities with A. locuta-pollinis (LC11683, ex-holotype) (accession nos. MF939595, MF939622, MF939616), and LSU data missing. Phylogenetic analyses using maximum likelihood showed a 92% bootstrap support value in a clade with A. locuta-pollinis (Fig 2). Eight healthy plants (2-year-old) were used for the pathogenicity test. Culms of four healthy bamboos were wounded via sterile double-edged blade and sprayed with conidial suspension (105 conidia/ml) prepared from 4-week-old cultures that were incubated on PDA at 25℃. The other four bamboos were sprayed with sterile distilled water as controls. Inoculated plants were placed in a growth chamber (25℃, 90% relative humidity, 12-h photoperiod). About 60 days later, necrotic patches similar to those observed in the field were found on the inoculated culms, and no symptoms were observed on the controls. The pathogen was reisolated from the diseased culms with identical morphology as previously described. To our knowledge, this is the first report of culm blight on P. aureosulcata 'Spectabilis' caused by A. locuta-pollinis. The risk of this pathogen needs further evaluation, and effective control measures should be taken.
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