Two new species of Ganoderma (Ganodermataceae, Basidiomycota) from Southwest China.

Ganoderma is a large and diverse genus containing fungi that cause white rot to infect a number of plant families. This study describes G.phyllanthicola and G.suae as new species from Southwest China, based on morphological and molecular evidence. Ganodermaphyllanthicola is characterized by dark brown to purplish black pileus surface with dense concentric furrows, pale yellow margin, irregular pileipellis cells, small pores (5-7 per mm) and ellipsoid to sub-globose basidiospores (8.5-10.0 × 6.0-7.5 µm). Ganodermasuae is characterized by reddish brown to oxblood red pileus surface and lead gray to greyish-white pore surface, heterogeneous context, wavy margin and almond-shaped to narrow ellipsoid basidiospores (8.0-10.5 × 5.0-7.0 µm). The phylogeny of Ganoderma is reconstructed with multi-gene sequences: the internal transcribed spacer region (ITS), the large subunit (nrLSU), translation elongation factor 1-α gene (TEF-1α) and the second subunit of RNA polymerase II (RPB2). The results show that G.suae and G.phyllanthicola formed two distinct line-ages within Ganoderma. Descriptions, illustrations and phylogenetic analyses results of the two new species are presented.

First report of large-berry coffee (Coffea liberica) anthracnose caused by Colletotrichum kahawae in China.

Large-berry coffee (Coffea liberica) is one of the three cultivated coffee species and a precious breeding germplasm in China (Yan et al, 2019). Anthracnose is a damaging epidemic disease on coffee worldwide (Mohammed et al. 2015). Between June and September 2022, anthracnose was observed on coffee plants in Puer area, Yunnan, China and disease incidence (% plants diseased) of 8.5%-28.2% was recorded in the field. The disease symptoms were observed at all growth stages. Lesions on leaves were circular or oval, with a white to gray central zone outlined by a brown margin and surrounded by a chlorotic halo, Φ5.1-18.5 mm; some lesions extended and coalesced later to form large, blighted areas, leading to complete leaf senescence, defoliation and bare blighted branches on heavily infected trees. The spots on coffee berries were oval or fusiform, sunken and brown-black; diseased berries became gray-black and dried-out but remained on the tree. Leaves with typical anthracnose lesions were collected from fields in Simao ( 22.07°E,100.98°N) to isolate the pathogen. Leaf pieces (5×5mm) from the lesion margin were cut, surface-sterilized with 75% ethanol and 2% NaClO, and cultured on PDA at 25°C. Three isolates with the same colony morphology were obtained by hyphal tip purification. Detached and intact leaves of 6-month coffee seedlings were inoculated with Φ5mm mycelial discs of the isolates. Anthracnose lesions developed on the inoculated leaves, with all 3 isolates, 7d after incubation in a growth chamber (25°C, > 90% RH and lighting 8 h/d at 11000 lux). Pathogens with the same colony morphology as those of the original isolates were re-isolated from the infected tissues of inoculated leaves, thus fulfilling Koch's Postulates. The ITS sequence (PP550861) for the isolate was PCR-amplified and Blast-n analyses showed 100 % (554/554bp) identity to Colletotrichum kahawae LWTJ01; so they were the same population and coded as KFTJ02. The actin (ACT), calmodulin(CAL), glyceraldehydes-3-phosphate dehydrogenase (GAPHD) and histone 3 (HIS3) genes (Qiu et al. 2020) were amplified from one of KFTJ02 isolates, sequenced and deposited in NCBI GenBank (OR842543, OR842544, OR842545 & OR842546). A phylogenetic tree was generated based on the concatenated sequences of the four genes and those of related Colletotrichum spp. using MEGA 6.0 and KFTJ02 clustered in the same clade with C. kahawae IMI319418 on the tree (Bootstrap sup.=88%). When cultured at 25°C on PDA for 7 days, its colonies were near round or ovoid, gray-white, contoured, Φ73.2-80.1 (76.2±2.3)mm or growth rate 10.2-11.1(8.1) mm/d (n=10). The hyphae were hyaline, septated, branching at near right angles. Conidial masses formed 14 days after incubation. The conidia were elliptical, hyaline, monocellular, 10.2-15.5 (12.7±1.06)×3.8-5.2 (4.3±0.52) µm (n=50). The appressoria were black-brown, oval or irregular, 7.8-9.3 (8.5±0.81)µm (n= 50). These morphological characteristics were consistent with those of C. kahawae (Bridge et al, 2008). Therefore, KFTJ02 was identified as C. kahawae, which has been found to infect Camellia oleifera, Areca catechu and Ficus microcarpa (Wei et al, 2023; Zhang et al, 2020; Lin 2023). The coffee berry disease pathogen (C. kahawae) is a quarantine species which has not been recorded and so it is first reported on coffee crops in China. Results of the present study provide important references for further studies on this disease.

Potential Biocontrol Microorganisms Causing Attenuated Pathogenicity in Plasmopara viticola.

A phenomenon of pathogenicity attenuation of Plasmopara viticola was consistently observed during its subculture on grape. To clarify the causes of attenuated pathogenicity of P. viticola, culturable microbes were isolated from the P. viticola mass (mycelia, sporangiophores, and sporangia) in each generation and tested for their biocontrol efficacies on grape downy mildew (GDM). The results showed that the incidence of GDM decreased with the increase in the number of subculture times on both vineyard-collected leaves and grape leaves from in vitro-grown seedlings. The number of culturable microbial taxa on the surface of P. viticola decreased, whereas the population densities of four specific strains (i.e., K2, K7, P1, and P5) increased significantly with the increase in subculture times. Compared with the control, the biocontrol efficacies of the bacterial strain K2 reached 87.5%, and those of both fungal strains P1 and P5 reached 100.0%. Based on morphological characteristics and molecular sequences, strains K2, P1, and P5 were identified as Curtobacterium herbarum, Thecaphora amaranthi, and Acremonium sclerotigenum, respectively, and these three strains survived very well and multiplied on the surface of P. viticola. As the number of times P. viticola was subcultured increased, all three of these strains became the predominant strains, leading to greater P. viticola inhibition, attenuated P. viticola pathogenicity, and effective GDM biological control. To the best of our knowledge, this is the first report of C. herbarum and T. amaranthi having biological control activity against GDM.

First report of Colletotrichum siamense as a causal agent of anthracnose on wax apple (Syzygium samarangense) in China.

Wax apple (Syzygium samarangense) is an important fruit tree widely cultivated in China. Yield losses are usually serious due to different diseases among which anthracnose (Colletotrichum spp.) is one of the most damaging (He et al, 2019). This disease was found in Yunnan, China and an average incidence of 56.7% diseased leaves was recorded in21 orchards surveyed in July2021. The disease lesions on leaves were circular, angular or oval (7.2-15.6 mm), with whitish center and brown outer area surrounded by a yellow halo; irregular spots or blight areas formed later. It can also infect fruits forming pale-brown, circular and sunken spots before harvest and rot of stored fruits. Diseased leaves were sampled from orchards in Ximeng (N117.78oE39.89o) and Ninger (E101.04oN23.05o) counties of Yunnan for fungal isolation; three and five pure isolates were recovered from Ximeng (LWTJ1-LWTJ3) and Ninger (LB4-LB8) samples, respectively, by plating disinfested tissue (surface-sterilized with 2% NaClO3) on potato dextrose agar (PDA) followed by hyphal tip purification and incubation at 25oC. Two repeated tests following Koch's postulates were conducted to verify pathogenicity of the eight isolates. In each test, three healthy seedlings per isolate were sprayed with conidia suspenson (2.26×105cfu/mL) until runoff from leaves while control plants were sprayed with sterile water. The plants were kept in the dark at RH100 for 24 h in a black box and then in a growth chamber (28oC, RH>90% and lighting 12h/d). Detached fruits were inoculated with mycelial discs on the puncture-wound surface. Anthracnose symptoms developed on all seedlings and fruits inoculated with LWTJ2 or LB4 isolates, which were re-isolated from lesions of inoculated leaf/fruit, completing Koch's postulates. Control plants were healthy and symptomless. LWTJ2 and LB4 isolates were morphologically the same: the colonies on PDA were circular, pale-white, with cottony surface and readily forming orange conidium masses. The hyphae were hyaline, septate, branched mostly in near right angles. The conidia were hyaline, one-celled, smooth-walled, cylindrical with round ends, 9.8-17.5 (av.13.8) µm×4.4-6.5 (5.6) µm. The teleomorph was not observed in culture or on orchard trees. The morphological characters were consistent with those of C. siamense described by Weir et al (2012). The internal transcribed spacer region (ITS) was amplified from the two isolates by PCR and sequenced (1990) and were 545 bp in length (OL963924 & OL413460). BLAST analysis showed that both were 100% identical and they shared 99.08% identity with C. siamense WZ-365 from the ITS region (MN856443).The Tub2 (788 bp, ON637119) and Cal (768 bp, ON622249) genes (Weir et al, 2012) of LB4 were also obtained and they shared closest identity (99.45% & 100%) with those of C. siamense WZ-365 as well. Phylogenetic tree (neighbor-joining) analysis of the concatenated sequence of ITS, Tub2 and Cal genes of LB4 and those of related Colletotrichum spp. showed that LB4 clustered IN the same end-branch with C. siamense ICMP18578 (Bootstrap sup. = 98%). Thus, C. siamense was identified as the pathogen of wax apple anthracnose in Yunnan. It caused anthracnose on other crops as oranges and cacao (Azad et al, 2020). Also, C. fructicola and C. syzygicola were identified as pathogens of wax apple anthracnose in Thailand (Al-Obaidi et al, 2017). To our knowledge, this is the first report of C. siamense causing wax apple anthracnose in China.

Analogical Assessment of Train-Induced Vibration and Radiated Noise in a Proposed Theater.

This study presents the analogical assessment of the train-induced vibration and radiated noise in a proposed theater. The theater is to be constructed in a region with crowded metro lines, and the assessment is implemented in an analogical building with comparable structural type and metro condition. Prior to the assessment, the comparability of the analogical building with the theater is validated using the train-induced ground vibration. With the same horizontal distance from the metro line, the train-induced vibration level in the analogical building is 9 dB higher than that in the construction site of the theater. Such results indicate that the lack of soil layers may lead to a dramatic increase in train-induced vibration in the building. In the staircase of the analogical building, the train-induced radiated noise reached 55 dB (A), which is 10 dB (A) higher than the daytime allowable level. As the most important indicator, the noise rating number in the cinema of the analogical building is NR-43, which put forward an enormous challenge on the construction of the theater with a denoise demand of 23 dB. The analogical method applied in this study provides an effective and practical way for the assessment of train-induced vibration and radiated noise in proposed vibration-sensitive buildings. The assessment results that provide necessary reference and support for the anti-vibration design will help guarantee the stage effect of the theater.

First report of a new leaf blight disease of bletilla (Bletilla striata) caused by Curvularia reesii in China.

Bletilla striata is an important Chinese herbal plant grown widely in southwest China (Qian et al. 2021). Leaf blight was found on cultivated bletilla crops in Yunnan in 2021. The disease infected bletilla leaves and it was present in the field from April to November with the highest incidence (86% plants diseased) recorded in early September in Puer area. Foliar lesions were circular (Φ0.5-1.8 cm) or oval, with pale-gray center and narrow gray-brown outer area surrounded by a yellow halo. The lesions coalesced later to form large irregular spots or blighted areas on leaves. Symptomatic bletilla leaves were sampled from fields in Jiangcheng (E101.8672o, N22.5803o) and Simao (E109.7816o, N22.7891o) counties, Yunnan in July 2021. Seven fungal isolates were obtained from (BJ01-BJ04) and Simao samples (HBJ05-HBJ07) via lesion-tissue culture and hypha-tip purification on PDA medium. A pathogenicity test following Koch's Postulates (Grimms et al. 2006) was conducted using each isolate by inoculating 45-day old bletilla plant (n=30, Zihua cultivar) in a greenhouse through spraying hypha-spore suspension (3.25×104 CFU/mL) prepared with 14 d fresh DNA culture. Non-inoculated plants (n=30) were used as controls. The experiment was repeated once. The isolates BJ02 and HBJ06 (deposited in Yunnan Agric. Univ. Microbes Herbarium) were shown pathogenic to bletilla since similar lesions formed on seedlings 7 d post inoculation and pure fungal cultures with the same colony morphology as those of BJ02 and HBJ06 were re-isolated from leaf lesions 14 dpi. Isolates BJ02 and HBJ06 produced identical colony and conidium morphology after they were incubated at 25oC for 7 d on PDA. Colonies were circular, pale brown, Φ5.5-7.5cm, with villous surface and abundant aerial hyphae. Mycelia were septate, colorless, Φ3-4 µm and with acute-angled branches. Conidiophores developed from hyphae were erect, septate, pale-brown colored and 60-200 µm long. Conidia (produced scarcely and ripened slowly) were long-oval or petaloid, straight or slightly curved, brown, sized 28-45×10-14 µm. Most conidia were divided into 4 cells by 3 septa; the middle two were bigger than the basal and apex cells. Both BJ02 and HBJ06 were identified as Curvularia sp. based on their morphological characters (Tan et al. 2018). The rDNA-ITS, TEF1α and GAPDH genes (Tan et al. 2018) were amplified from these isolates with PCR (White et al. 1990) and sequenced. ITS sequences of the two isolates were both 574 bp (acc. no. OL587997 & OL336480) and 100% (574/574 bp) identical shown by blast comparison. Further blast analyses of ITS (574 bp, OL587997), TEF1α (532 bp, ON637120) and GAPDH (881 bp, ON637121) from isolate BJ02 showed that they were 99.27% (547/551 bp), 100% (842/842 bp) and 99.8% (507/508 bp) identical respectively with those of Curvularia reesii BRIP4358 (MH414907). The 3 genes of BJ02 were concatenated and phylogenic analysis (Tamura et al, 2013) of the concatenated sequence with those of Curvularia spp. showed that BJ02 was clustered with C. reesii BRIP4358 on the same end-branch of the tree with 100% confidence. Therefore, BJ02 and HBJ06 are the same species identified as Curvularia reesii and it is the pathogen causing bletilla leaf blight. C. reesii was first isolated from the air in Australia in 1963 and was named by Tan et al. in 2018. It has not been reported as a plant pathogen elsewhere. This is the first record of this fungus causing bletilla leaf blight in China. Keywords: Bletilla striata; leaf blight; Curvularia reesii; disease symptoms; pathogen morphology; multigene identification References (1) D.J. Grimes. Microbes, 1(5): 223-228, 2006. (2) L.H. Qian et al. Jiangshu Agric. Sci. 49(19): 64-71, 2021. (3) K. Tamura et al. Mol. Bio. & Evol. 30 (12): 2725- 2729, 2013. (4) Y. P. Tan et al. MycoKeys, 35: 1-25. 2018. (5) T.J. White et al. In: PCR Protocols: A Guide to Methods and Applications (eds. M.A. Innis et al.), Acad. Press, Inc. New York. 315-322, 1990.

Tobacco curly shoot virus Down-Regulated the Expression of nbe-miR167b-3p to Facilitate Its Infection in Nicotiana benthamiana.

Tobacco curly shoot virus (TbCSV) belongs to the genus Begomovirus of the family Geminiviridae, and causes leaf curling and curly shoot symptoms in tobacco and tomato crops. MicroRNAs (miRNAs) are pivotal modulators of plant development and host-virus interactions. However, the relationship between TbCSV infection and miRNAs accumulation has not been well investigated. The present study was conducted to analyze different expressions of miRNAs in Nicotiana benthamiana in response to the infection of TbCSV via small RNAs sequencing. The results showed that 15 up-regulated miRNAs and 12 down-regulated miRNAs were differentially expressed in TbCSV infected N. benthamiana, and nbe-miR167b-3p was down-regulated. To decipher the relationship between nbe-miR167b-3p expression and the accumulations of TbCSV DNA, pCVA mediation of miRNA overexpression and PVX based short tandem target mimic (STTM) were used in this study. It was found that overexpression of nbe-miR167b-3p attenuated leaf curling symptom of TbCSV and decreased viral DNA accumulation, but suppression of nbe-miR167b-3p expression enhanced the symptoms and accumulation of TbCSV. PRCP, the target gene of nbe-miR167b-3p, was silenced in plants using VIGS and this weakened the viral symptoms and DNA accumulation of TbCSV in the plants. Overall, this study clarified the effect of nbe-miR167b-3p on plant defense during TbCSV infection, and provided a framework to reveal the molecular mechanisms of miRNAs between plants and viruses.

Ss-Rhs1, a secretory Rhs repeat-containing protein, is required for the virulence of Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall-degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss-Rhs1, which is essential for the virulence of S. sclerotiorum. Ss-Rhs1 is believed to contain a typical signal peptide at the N-terminal and eight rearrangement hotspot (Rhs) repeats. Ss-Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss-Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss-Rhs1-silenced strains exhibited reduced efficiency in compound appressoria formation.