Genome analysis and classification of Xanthomonas bacteriophage AhaSv, a new member of the genus Salvovirus.

Xanthomonas phage AhaSv was isolated from lake water. Genome sequencing showed that its genome is a linear dsDNA molecule with a length of 55,576 bp and a G+C content of 63.23%. Seventy-one open reading frames (ORFs) were predicted, and no tRNAs were found in the genome. Phylogenetic analysis showed that AhaSv is closely related to members of the genus Salvovirus of the family Casjensviridae. Intergenomic similarity values between phage AhaSv and homologous phages were up to 90.6%, suggesting that phage AhaSv should be considered a member of a new species in the genus Salvovirus.

First Report of Powdery Mildew on Sophora flavescens Caused by Erysiphe diffusa in China.

Sophora flavescens (Fabaceae) is a deciduous subshrub which has been used in Chinese popular medicine for a long history (He et al. 2015). In June 2023, severe powdery mildew symptoms were observed on wild S. flavescens plants on Longwen hill of Guizhou Normal University, Guiyang, China. The incidence was approximately 80% among 100 S. flavescens plants observed. Almost all leaves were infected. Mycelia occurred on both adaxial and abaxial leaf surfaces, petioles, and stems, forming small-to-large patches. Hyphae were hyaline, 5 to 7 µm wide. Hyphal appressoria were solitary. Conidiophores were erect, straight to somewhat flexuous, and 45 to 120 µm long (n = 50). Foot cells were subcylindrical to slightly curved, followed by 2 to 3 shorter cells. Conidia formed singly, were ovoid to cylindrical, 26 to 42 × 12 to18 µm (n = 50). Based on these morphological characteristics, the powdery mildew fungus was tentatively identified as Erysiphe diffusa (Braun and Cook 2012). To confirm the identification, the ribosomal DNA internal transcribed spacer (ITS) and the ribosomal large subunit (LSU) region were amplified and sequenced using primer pairs ITS1/ITS4 (White et al. 1990) and NL1/NL4 (Ziemiecki et al. 1990), respectively. The obtained 647-bp ITS sequence (GenBank accession no. PP130131) displayed 100% identity with the ITS sequences of E. diffusa. The obtained 618-bp LSU sequence (GenBank accession no. PP693303) displayed 100% identity with the ITS sequences of E. diffusa (MT325922 and MT628019) and E. manihoticola (MT106658 and MT106660). Using a phylogenetic tree based on the combined ITS-LSU data, the isolate was grouped in a clade with the E. diffusa strain (GenBank accession no. LC777871). To fulfill Koch's postulates, leaves of three healthy potted S. flavescens plants were inoculated by gently pressing with diseased leaves. Non-inoculated plants were used as controls. All plants were incubated in a greenhouse at 25 ± 2°C, 80% relative humidity. After 15 days, typical powdery mildew symptoms were observed on the inoculated plants, whereas no symptoms were found on the control plants. The reisolated fungus from the inoculated S. flavescens was morphologically identical to that on naturally diseased plants, and the ITS sequence of the reisolated fungus showed 100% identity with PP130131. As the causal fungus of soybean powdery mildew, E. diffusa is known to infect papaya and other legumes, including Lens culinaris and Mimosa caesalpiniifolia (Attanayake et al. 2009; Luz et al. 2019). Particularly, E. diffusa has been previously reported to infect S. flavescens in the United Kingdom (Jones and Baker 2007; Bradshaw et al. 2023), but this is the first report of S. flavescens powdery mildew caused by E. diffusa in China. This work further expands the geographical range of E. diffusa-infected S. flavescens plants.

First Report of Powdery Mildew on Quercus fabri and Q. robur Caused by Erysiphe quercicola in China.

Quercus (Fagaceae) is a genus of ecologically and economically important shrub and tree species (Yin et al. 2018). In April 2022, powdery mildew symptoms were observed on Quercus fabri and Quercus robur leaves on Longwen hill, Guizhou Normal University, Guiyang, China. The incidence was 30% (Q. fabri, n = 50) and 20% (Q. robur, n = 30), respectively. Powdery mildew fungi from these two Quercus species shared similar morphological characteristics. Mycelia occurred on adaxial and abaxial leaf surfaces, forming small to large patches; hyphae were hyaline, 3-7 µm wide; hyphal appressoria were lobed to multilobed, solitary; conidiophores were erect, straight, 36-80 µm long (n = 30); foot cells were followed by 1-2 shorter cells; conidia formed singly, obovoid to ellipsoid, 24-38 × 12-27 µm (n = 50), without fibrosin bodies; no chasmothecia were observed. Based on these characteristics, powdery mildew fungi on both Q. fabri and Q. robur were identified as Erysiphe quercicola (Takamatsu et al. 2007). To confirm the identification, ribosomal DNA internal transcribed spacer (ITS) sequences of two fungal samples from Q. fabri and Q. robur were separately amplified and sequenced using primer pair ITS1/ITS4 (White et al. 1990). The obtained ITS sequences (GenBank accession nos. QR414372 and QR414373, respectively) shared 100% identity, and 99.38-99.84% identity with diverse ITS sequences of E. quercicola (Takamatsu et al. 2015). In a phylogenetic tree based on ITS sequences of Erysiphe species (Takamatsu et al. 2007), QR414372 and QR414373 were grouped in a clade with ITS sequences of E. quercicola. To fulfil Koch's postulates, leaves of three healthy potted Q. fabri plants and three healthy potted Q. robur plants were inoculated by gently pressing diseased Q. fabri and Q. robur leaves onto healthy leaves. Non-inoculated healthy Q. fabri and Q. robur plants served as controls. All plants were incubated in a greenhouse at 25 ± 2°C with 80% relative humidity. Typical powdery mildew symptoms were observed on all inoculated plants 15 days after inoculation, whereas no symptoms were observed on control plants. Fungi separately reisolated from inoculated Q. fabri and Q. robur were morphologically identical to those on their originally diseased plants, and ITS sequences of reisolated fungi shared 100% identity with QR414372 and QR414373. E. quercicola has previously been reported to infect Quercus species, including Q. robur in Australia, Q. crispula, Q. phillyraeoides and Q. serrata in Japan, and Q. phillyraeoides in Korea (Lee et al. 2011). In China, Q. fabri and Q. robur may be infected by E. alphitoides and E. hypophylla, respectively (Zheng et al. 1987). To our knowledge, this is the first report of powdery mildew caused by E. quercicola on Q. fabri and Q. robur in China. This work provides a foundation to protect Quercus plants against this fungal pathogen.

Gene redundancy and gene compensation of insulin-like peptides in the oocyte development of bean beetle.

Bean beetle (Callosobruchus maculatus) exhibits clear phenotypic plasticity depending on population density; However, the underlying molecular mechanism remains unknown. Compared to low-density individuals, high-density individuals showed a faster terminal oocyte maturity rate. Four insulin-like peptide (ILP) genes were identified in the bean beetle, which had higher expression levels in the head than in the thorax and abdomen. The population density could regulate the expression levels of CmILP1-3, CmILP2-3, and CmILP1 as well as CmILP3 in the head, thorax, and abdomen, respectively. RNA interference results showed that each CmILP could regulate terminal oocyte maturity rate, indicating that there was functional redundancy among CmILPs. Silencing each CmILP could lead to down-regulation of some other CmILPs, however, CmILP3 was up-regulated in the abdomen after silencing CmILP1 or CmILP2. Compared to single gene silencing, silencing CmILP3 with CmILP1 or CmILP2 at the same time led to more serious retardation in oocyte development, suggesting CmILP3 could be up-regulated to functionally compensate for the down-regulation of CmILP1 and CmILP2. In conclusion, population density-dependent plasticity in terminal oocyte maturity rate of bean beetle was regulated by CmILPs, which exhibited gene redundancy and gene compensation.

Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Characteristics and whole-genome analysis of a novel Pseudomonas syringae pv. tomato bacteriophage D6 isolated from a karst cave.

Pseudomonas syringae is a gram-negative plant pathogen that infects plants such as tomato and poses a threat to global crop production. In this study, a novel lytic phage infecting P. syringae pv. tomato DC3000, named phage D6, was isolated and characterized from sediments in a karst cave. The latent period of phage D6 was found to be 60 min, with a burst size of 16 plaque-forming units per cell. Phage D6 was stable at temperatures between 4 and 40 °C but lost infectivity when heated to 70 °C. Its infectivity was unaffected at pH 6-10 but became inactivated at pH ≤ 5 or ≥ 12. The genome of phage D6 is a linear double-stranded DNA of 307,402 bp with a G + C content of 48.43%. There is a codon preference between phage D6 and its host, and the translation of phage D6 gene may not be entirely dependent on the tRNA library provided by the host. A total of 410 open reading frames (ORFs) and 14 tRNAs were predicted in its genome, with 92 ORFs encoding proteins with predicted functions. Phage D6 showed low genomic similarity to known phage genomes in the GenBank and Viral sequence databases. Genomic and phylogenetic analyses revealed that phage D6 is a novel phage. The tomato plants were first injected with phage D6, and subsequently with Pst DC3000, using the foliar spraying and root drenching inoculum approach. Results obtained after 14 days indicated that phage D6 inoculation decreased P. syringae-induced symptoms in tomato leaves and inhibited the pathogen's growth in the leaves. The amount of Pst DC3000 was reduced by 150- and 263-fold, respectively. In conclusion, the lytic phage D6 identified in this study belongs to a novel phage within the Caudoviricetes class and has potential for use in biological control of plant diseases.

First Report of Rust Disease on Alcea rosea caused by Puccinia modiolae in China.

Alcea rosea, belonging to the Alcea genus in the Malvaceae family, originated from China, but it is now grown worldwide. A. rosea has been widely used in traditional Chinese medicine to alleviate constipation, pain, swelling, and sores. In February 2023, typical symptoms of fungal infection were observed on A. rosea at Guizhou Normal University in Guiyang, Guizhou Province, China. The disease incidence was over 90% (n = 100) for the surveyed A. rosea plants, and the disease severity range from 30% to 90%. The initial symptoms of A. rosea rust were the appearance of chlorotic spots on the leaves. Subsequently, numerous reddish to dark-brown erumpent pustules (telia) were observed. Gradually, the entire plant was covered by rust and the center of each lesion turned brown, necrotic, and ruptured over times, eventually causing defoliation. Voucher specimens of infected A. rosea leaves as representative samples have been deposited at Guizhou Normal University (GNU2023LS008). Telia are round in shape, mostly aggregated in mass, with a diameter of 0.28-0.78 mm (0.46 mm, n = 20). They range in color from reddish-brown to dark brown, and are mainly hypophyllous but occasionally formed on the adaxial leaf surface. The teliospores are fusoid with dimensions of 31.3-93.8 × 10.9-21.3 µm (57.5 × 15.1 µm average, n = 50), hyaline or yellowish to light-brown in color, mostly two-celled, with a smooth wall (1.5-3.0 µm) and a thickened apex (3.0-9.0 µm). However, teliospores which are one-, three-, or four-celled with a notch at the apex, are rarely observed. The morphological characteristics of host symptoms and teliospores were similar to those of Puccinia modiolae (Aime and Abbasi 2018; Albu et al. 2019). For phylogenetic analysis, genomic DNA was extracted from the teliospores of infected leaves. To confirm the species-level identification, PCR was performed on the extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS) and large subunit (LSU) regions using primer pairs ITS1/ITS4 (Schoch et al. 2012) and NL1/NL4 (Ziemiecki et al. 1990), respectively. The resulting ITS DNA sequence (GenBank accession no. OR607960) showed 100% identity with P. modiolae sequences (OP369291.1), when the query coverage was 100%. The LSU DNA sequence obtained (OR607961.2) shared 99.85% similarity with P. modiolae (MK458702.1). A phylogenetic tree was constructed using MEGA7.0 and the maximum likelihood method based on the ITS and LSU sequences. The fungal isolates collected in this study and several reference sequences of P. modiolae were grouped within a clade that included the isolates reported on A. rosea in Korea (Ryu et al. 2023), with 100% bootstrap support. Pathogenicity testing was conducted by gently pressing spore powder of naturally diseased leaves onto young leaves of three healthy A. rosea plants, with three noninoculated healthy plants serving as controls. The inoculated and noninoculated plants were kept in a growth chamber at the 26°C with a 12 hour light/dark cycle and 80% humidity. After 2 weeks, all inoculated A. rosea plants showed characteristic disease symptoms of rust infection and telia of P. modiolae, while control plants remained symptomless. The pathogen was identical to that observed on the original diseased leaves. The study results indicate that the causal fungus responsible for the disease is P. modiolae, which has been previously reported on Malvaceae plants (Farr and Rossman 2022). To the best of our knowledge, this is the first report of P. modiolae on A. rosea in China. This study will contribute to an increased understanding of the host range of Puccinia modiolae.

First Report of Powdery Mildew on Viburnum chinshanense Caused by Erysiphe pseudoviburni in China.

Viburnum chinshanense, a deciduous shrub in the family Caprifoliaceae, is a dominant tree distributed mainly in the North-Central and South-Central regions of China (Zhu et al. 2023). Because of its lush white flowers and vibrant red fruits, V. chinshanense is used widely as ornamental tree in China. In May 2022, severe powdery mildew symptoms were observed on V. chinshanense on the Huaxi Campus of Guizhou Normal University, Guiyang, China. The incidence was approximately 75% among 80 V. chinshanense plants observed. White mycelia were present on both adaxial and abaxial leaf sides, but not on fruits, petioles, or stems. Infected leaves showed slight chlorosis and twisting. The mycelia were amphigenous, forming small-to-large patches, often sparse on the upper leaf surface, but mostly confluent on the lower leaf surface. Hyphae were hyaline, 4-7 µm wide. Hyphal appressoria were lobed to multilobed, in opposite pairs or solitary. Conidiophores were erect, straight, or somewhat flexuous, 60-130 µm long (n = 30). Foot cells were subcylindrical to slightly curved-sinuous at the base, 20-40 × 6-10 µm (n = 30) in size, followed by 1-3 shorter cells. Conidia formed singly, occasionally two to three in a chain. Conidia were ellipsoid to ovoid, cylindrical, and 24-40 × 16-20 µm (n = 50). No fibrosin bodies were observed on the conidia. Chasmothecia were subglobose, 56-115 µm in diameter. The appendages were 35-70 µm long. Based on these morphological characteristics, the powdery mildew fungus was identified as Erysiphe pseudoviburni (Bradshaw et al. 2020). To confirm the identification, the ribosomal DNA internal transcribed spacer (ITS) and the ribosomal large subunit (LSU) region were amplified and sequenced using the ITS1/ITS4 primer pair (White et al. 1990) and the NL1/NL4 primer pair (Ziemiecki et al. 1990), respectively. The obtained 643-bp ITS sequence (GenBank accession no. ON729292) had 99.84% identity with E. pseudoviburni strains KUS-F27310 (MN431595) and MUMH0001 (LC009904). The obtained 593-bp LSU sequence (ON729293) had 99.83% identity with E. pseudoviburni (LC009904 and MN431595). Based on the phylogenetic analysis of the combined ITS and LSU dataset (Bradshaw et al. 2020), the isolate (GZVD-1) was grouped in a clade with the E. pseudoviburni strains KUS-F27319, KUS-F27310, and MUMH0001. To fulfill Koch's postulates, leaves of three healthy potted V. chinshanense plants were inoculated by gently pressing with diseased leaves. Non-contact plants were used as controls. All plants were incubated in a greenhouse at 25 ± 2°C, 80% relative humidity. Similar powdery mildew symptoms were observed on the inoculated plants 12 days after inoculation, whereas the control plants remained symptomless. The reisolated fungus from the inoculated plants was morphologically identical to that on originally diseased plants. ITS and LSU sequences of the reisolated fungus showed 100% identity with ON729292 and ON729293, respectively. E. pseudoviburni has previously been reported to infect some Viburnum species, including V. sieboldii in Japan (Takamatsu et al. 2015) and V. odoratissimum in South Korea (Bradshaw et al. 2020). To the best of our knowledge, this is the first report of powdery mildew caused by E. pseudoviburni on V. chinshanense in China. This work expands the known host range of E. pseudoviburni in the Viburnum genus.

First Report of Powdery Mildew on Berchemia floribunda Caused by Erysiphe berchemiae in China.

The genus Berchemia (family Rhamnaceae), a group of climbing plants, is mainly distributed in Asia, Africa, and South America. Berchemia plants are widely used in traditional medicine in some Asian countries (Inoshiri et al. 1987). For example, in Japan, B. racemosa (synonym B. floribunda) is used for the treatment of gallstones, liver diseases, neuralgia, and stomach cramps, and in China, B. floribunda is used for the treatment of rheumatism and lumbago. In August 2022, typical powdery mildew symptoms were observed on wild B. floribunda plants in Huaxi District, Guiyang, Guizhou Province, China. The incidence was approximately 60% among 100 B. floribunda plants observed outdoors. White colonies almost entirely covered on both adaxial and abaxial surfaces of all leaves on symptomatic plants. Infected leaves appeared curled or chlorotic, infection occasionally leading to defoliation. To describe the pathogen morphologically, fungal samples were collected from two individual B. floribunda plants and microscopically characterized. In these samples, hyphae were flexuous to straight, branched, septate, 3-6 µm wide, with lobed hyphal appressoria. Conidiophores were erect, flexuous to straight, and 50-160 µm long (n = 30). Foot cells were subcylindrical to slightly curved-sinuous at the base, 20-40 µm long (n = 30), followed by 2-3 shorter cells. Conidia formed singly, occasionally 2-3 in a chain. Conidia were ellipsoid to ovoid, 20-42 × 12-18 µm (n = 50), without fibrosin bodies. Chasmothecia were not found. For molecular identification, the ribosomal DNA internal transcribed spacers (ITSs) of the two fungal samples were amplified and sequenced using the ITS1/ITS4 primer pair (White et al. 1990). The obtained 649-bp ITS sequences (GenBank accession nos. OR414364 and OR414365, respectively) shared 100% identity, and they showed 99.52% identity with the ITS sequence (GenBank accession no. LC009934) of Erysiphe berchemiae. Phylogenetic analysis grouped OR414364 and OR414365 in a clade with LC009934. Based on morphological and molecular characteristics, the powdery mildew fungus from B. floribunda was identified as E. berchemiae (Braun and Cook 2012). The voucher specimen (accession no. GZNU-BFEE/0820/2022) were deposited at the School of Life Sciences, Guizhou Normal University. Pathogenicity was assessed by gently pressing naturally diseased leaves of B. floribunda onto leaves of three healthy potted 1-year-old B. floribunda plants. Three non-inoculated healthy plants were used as controls. The plants were incubated in a greenhouse at 25 ± 2°C, 80% relative humidity. Similar powdery mildew symptoms were observed on the inoculated plants 9 days after inoculation, whereas the control plants remained symptomless. The reisolated fungus from inoculated leaves was morphologically identical to that observed on the original diseased leaves, and the ITS sequence of the reisolated fungus shared 100% identity with OR414364 and OR414365, fulfilling Koch's postulates. E. berchemiae has previously been described as a powdery mildew pathogen on B. yunnanensis (Chen et al. 1987) and B. kulingensis (Chen 1993) in China and B. racemosa (synonym B. floribunda) in Japan (Braun and Cook 2012; Takamatsu et al. 2015), but this is the first report of E. berchemiae causing disease of B. floribunda in China. This work suggests that E. berchemiae is an important pathogen of Berchemia plants, at least for some species in the genus Berchemia.

First Report of Powdery Mildew on Photinia × fraseri Caused by Podosphaera leucotricha in Guizhou Province, China.

The evergreen shrub Photinia × fraseri is a Photinia glabra × Photinia serrulata hybrid belonging to the family Rosaceae that is widely used in ornamental landscaping. In March 2022, severe powdery mildew symptoms were observed on shrubs of Photinia × fraseri in Huaxi University Town, Guiyang, Guizhou Province, China. All observed Photinia × fraseri plants in the green belts of both roads and parks in University Town showed powdery mildew symptoms. Almost all young branches of each Photinia × fraseri individual was infected. Powdery mildew colonies covered twig tips entirely, including the stems, petioles, and the adaxial and abaxial surfaces of leaves. Infected leaves were rolled up and had irregular, dark red spots. Fungal hyphae were straight to flexuous, branched, septate, 3 to 6 µm in width, and had nipple-shaped appressoria. Conidiophores were erect, straight or somewhat flexuous, and measured 90 to 300 µm × 7 to 10.5 µm (n = 30). Foot-cells were cylindrical or subcylindrical, straight or somewhat flexuous, and measured 25 to 50 × 7 to 9.5 µm (n = 30). Foot-cells were followed by one to two shorter cells, these being 10 to 16 × 7 to 9.5 µm in size (n = 50). Shorter cells were followed by one to six conidia (most often five conidia). Conidia formed in chains, ellipsoid to ovoid in shape, having dimensions of 22.5 to 30 × 12.5 to 16 µm (n = 50), and containing fibrosin bodies. No chasmothecia (fruiting bodies) were observed. Based on these morphological characteristics, the pathogen was identified as Podosphaera leucotricha (Ellis & Everh.) E.S. Salmon (Braun & Cook 2012). To confirm this species-level identification, the ribosomal DNA internal transcribed spacer (ITS) was amplified using the primers ITS1/ITS4 (White et al. 1990). The resulting sequence was deposited in GenBank (Accession No. ON325389). When the query coverage is 100%, the obtained ITS sequence showed 99.8% identity with P. leucotricha (AB027231, MT180425, MZ298746, KX842350, and KY661036) and 100% with P. leucotricha (HM242221, KY661017, KY661028, KY661050, KY661076, KR048110, MW364489, MW364490, MZ343479, OM022112, ON073894, and ON325389), respectively. Based on the ITS sequences of Podosphaera spp., phylogenetic tree was constructed with MEGA7.0 using the Maximum Likelihood method. The ML analysis supported our isolate's putative identification as P. leucotricha. To fulfill Koch's postulates, pathogenicity testing was conducted by gently pressing naturally diseased leaves onto young leaves of three healthy, potted 1-year-old Photinia × fraseri plants; three non-inoculated healthy plants served as control. Powdery mildew symptoms were observed on 100% inoculated Photinia × fraseri plants after 12 days (in a growth chamber at 21°C under a 12 h/12 h light/dark cycle), whereas the control plants remained symptomless. The powdery mildew colonies on inoculated leaves were morphologically identical to those observed on the original diseased leaves. It is known that P. leucotricha causes powdery mildew on Photinia × fraseri in Italy (Garibaldi et al. 2005). Moreover, this fungus reportedly infected Photinia serrulata in New Zealand, Ukraine, Italy, the United States, Japan, and in East China's Shandong Province (Liang et al. 2012). To the best of our knowledge, this is the first report of powdery mildew caused by P. leucotricha on Photinia × fraseri in Southwest China's Guizhou Province. This finding is significant as P. leucotricha is the causal agent of powdery mildew on apple and pear (Strickland et al. 2021). The occurrence of said disease on Photinia × fraseri could pose a potential disease threat to these fruit crops if nearby ornamental shrubs were able to act as reservoirs for the fungus, and a means to escape agricultural management efforts.

First Report of Leaf Blight on Dumasia villosa Caused by Boeremia exigua in China.

Dumasia (Fabaceae, tribe Phaseoleae, subtribe Glycininae), a genus of trifoliate vines, is widely distributed in tropical and subtropical regions of Asia and Africa (Pan & Zhu 2010). In October 2021, lesions were observed on Dumasia villosa leaves on Longwen mountain of Guizhou Normal University, Guiyang City, Guizhou Province, China. The incidence of leaf blight on observed D. villosa leaves was 10%. All necrotic spots were close to insect-feeding sites. Necrotic spots were grey or black, and circular (2 to 10 mm in diameter) or irregular (2 to 20 mm long) in shape. Stems and pods showed no disease symptoms. Pathogen isolation was conducted following surface sterilization with ethanol. After growth for 5 days on potato dextrose agar in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, colonies were 5.2 to 5.6 cm in diameter, olivaceous grey in colour, and sparse, white, aerial mycelia with irregular margins were evident. Conidia were hyaline, ellipsoidal to oblong, mostly 1-septate, and occasionally aseptate with dimensions ranging from 3.5 to 7.0 × 1.5 to 3.5 µm (n = 30). To confirm the species of the isolate, ribosomal DNA internal transcribed spacer (ITS) and ß-tubulin (tub2) genes were amplified and sequenced using primers ITS1/ITS4 and Btub2Fd/Btub4Rd (White et al. 1990), respectively. The obtained 529 bp ITS sequence (GenBank accession no. OL872186) shares 99.61% identity with the sequences of Boeremia exigua (MF662797 and GU395499). The obtained 299 bp tub2 sequence (OM830712) shares 100% identity with the sequence of B. exigua (KR653201). Based on morphology and DNA sequence analysis, the isolate was identified as B. exigua. To fulfil Koch's postulates, healthy leaves of 8-week-old D. villosa plants (n = 6) were wounded with a sterilized hypodermic needle and inoculated with 2 µL of a conidial suspension (106 conidia/mL). Six plants were inoculated with 2 µL of sterile distilled water as controls. After 3 days in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, dark spots were only present on conidia-inoculated leaves, while controls remained healthy. Boeremia exigua was reisolated from disease spots and confirmed using the same morphological and molecular methods described above. To our knowledge, this is the first report of leaf blight on D. villosa caused by B. exigua, although this fungus has been reported to infect leguminous plants, including field pea (Li et al. 2012), white clover (Wang et al. 2020), and soybean (Schaffrath et al. 2020). In China, B. exigua has also been reported to infect other plants, such as walnut (Cai et al. 2021; Wang et al. 2021) and Japanese ginseng (You et al. 2015). This identification suggests that D. villosa could be a potential reservoir for the pathogen affecting other leguminous crops that might be economically important.

First Report of Powdery Mildew on Rosa cymosa Caused by Podosphaera pannosa in Guizhou Province, China.

Rosa cymosa is a traditional Chinese medicine and an ornamental plant in China (Fan et al. 2020). In April 2022, powdery mildew symptoms were observed on R. cymosa in Guiyang, Guizhou Province, China. The incidence was approximately 5% among all observed one hundred R. cymosa plants. On average, 20% of the twig tips per diseased R. cymosa plant were affected. Powdery mildew colonies covered the adaxial and abaxial surfaces of leaves. Infected young leaves rolled up along the main vein. Stems and mature leaves occasionally had signs of powdery mildew. The hyphae were straight to flexuous, branched, septate, 3 to 6 µm in width. Conidiophores were erect, straight or somewhat flexuous, and 100 to 235 µm long. Foot cells (n = 30) were cylindrical or subcylindrical, straight or somewhat flexuous, and measured 20 to 48 µm (length) × 4.5 to 5.5 µm (width). Foot cells were followed by one to two shorter cells (n = 30) that measured 8 to 12 µm (length) × 4.5 to 5.8 µm (width). The shorter cells were followed by a chain of four to eight conidia. The conidia (n = 50) were hyaline, cylindrical to ovoid, with fibrosin bodies, and measured 20 to 28 µm (length) × 10.5 to 16.5 µm (width). No chasmothecia were observed on the surface of diseased leaves. For molecular identification, the ribosomal DNA internal transcribed spacer (ITS) was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). The obtained 508-bp ITS sequence was deposited in GenBank (Accession No. ON316871). The subsequent phylogeny grouped the ITS sequence within a clade of Podosphaera pannosa sequences. Based on both morphological and phylogenetic characteristics, the powdery mildew pathogen was identified as P. pannosa (Braun et al. 2002). The voucher specimen (Accession No. GZNU-RCPP/0804/2022) were deposited at the School of Life Sciences, Guizhou Normal University, China. Pathogenicity was assessed by gently pressing naturally diseased leaves onto young leaves of three healthy, potted 2-year-old R. cymosa plants, with three non-inoculated plants as controls. Powdery mildew symptoms were observed on all inoculated plants after incubation for 10 days at 21°C, 75% relative humidity, and 12 h/12 h light/dark cycle in a greenhouse. The control plants remained symptomless. The re-isolated powdery mildew colonies on inoculated leaves were morphologically identical to those observed on the original diseased leaves, fulfilling Koch's postulates. Podosphaera pannosa has been described as the most frequent species causing powdery mildew on the Rosaceae family, particularly on Rosa spp. and Prunus spp. (Hubert et al. 2012; Félix-Gastélum et al. 2014). The occurrence of powdery mildew on R. cymosa caused by P. pannosa could pose a potential disease threat to other Rosa crops or Prunus spp.

Arthrobacter cavernae sp. nov., a novel actinobacterium isolated from sediment of karst cave.

A novel, Gram-stain-positive, aerobic, non-endospore-forming, non-motile and rod-shaped bacterium designated PO-11T was isolated from sediment of karst cave collected in Libo county, Guizhou Province, PR China. The isolate grew optimally on R2A agar at 25 °C, pH 8.0 and with 0.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences showed that PO-11T belonged to the genus Arthrobacter and was most closely related to Arthrobacter methylotrophus TGAT (98.3 % sequence similarity), Arthrobacter alkaliphilus LC6T (97.7 %) and Arthrobacter ramosus CCM1646T (97.1 %). Genome sequencing revealed a genome size of 4 073 119 bp and the genomic DNA G+C content was 66.16 mol%. Its DNA-DNA relatedness values with A. methylotrophus TGAT, A. alkaliphilus LC6T and A. ramosus CCM1646T were 23.0, 22.9 and 23.2 %, respectively. The main fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major respiratory quinone was MK-9(H2). The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, glycolipid, phosphatidylethanolamine, phosphatidylinositol and unidentified lipids. Thus, based on phylogenetic and phenotypic and chemotaxonomic data, strain PO-11T represents a novel species of the genus Arthrobacter, for which the name Arthrobacter cavernae sp. nov. is proposed. The type strain is strain PO-11T (=CCTCC AB 2021070T=LMG 32459T).

First Report of Colletotrichum boninense Causing Anthracnose on Aucuba japonica in Guizhou Province of China.

Aucuba japonica is a plant with colorful leaves that is widely used in landscaping. Due to its shade tolerance and cold resistance (Li et al. 2013), A. japonica has become a dominant plant species in Guiyang city, China. From 2018 to 2021, an anthracnose disease was observed on A. japonica in Huaxi District (26°23'03'' N, 106°37'58'' E), Guiyang, Guizhou Province. The incidence of anthracnose in newly planted A. japonica reached 90%, resulting in a mortality rate of 30%. This has become a major disease for A. japonica in Guiyang. Typical symptoms include faded spots that initially appear at the infection center, followed by black-brown lesions with irregular edges. At the latter stage, slightly raised black spots are seen arranged in a wheel pattern. In severe cases, diseased leaves fall off. To identify the pathogen, leaf pieces (5 mm × 3 mm) containing symptomatic tissue with healthy margins were excised. The pieces were sterilized with 75% ethanol for 1 min, rinsed three times with sterile water, and cultured on potato dextrose agar (PDA) in Petri dishes at 26°C. White to cream colonies were developed after growth on PDA for 2 days. Mycelial growth ranged from 4.8 to 5.0 mm/day. Conidia were cylindrical, obtuse apex, and protruding base, with dimensions of 18.5 to 20.0 × 7.0 to 9.0 µm (n = 20). Conidial length:breadth ratio was 2-3 (n = 20). Acervuli and sclerotia had not been seen on PDA. To confirm the species of the isolate, PCR was performed on extracted DNA to amplify the ribosomal DNA internal transcribed spacer (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the calmodulin (CAL) gene, and the large subunit (LSU) region by using the primer pairs ITS1/ITS4 (Schoch et al. 2012), GDF1/GDR1 (Guerber et al. 2003), CAL228F/CAL737R (Carbone & Kohn 1999), and NL1/NL4 (Ziemiecki et al. 1990), respectively. The obtained 553 bp ITS sequence (Genbank accession no. MZ424503) showed 99.82% similarity with several C. boninense sequences (MN429163, MN542218, KM520024, and KM520014). The obtained 233 bp GAPDH sequence (OM643394) showed 99.07% similarity with C. boninense (MT602277, GQ221769, MK544890, and MK544888). The obtained 763 bp CAL sequence (OM313386) showed 99.32% similarity with C. boninense (MK569127). The obtained 584 bp LSU sequence (OK513272) showed 99.83% similarity with C. boninense (MH876452, MH877053, and MH876450). Phylogenic trees based on the sequences of ITS, GAPDH, and CAL (Damm et al. 2012), the species of isolate was confirmed as C. boninense. To confirm pathogenicity and to fulfill Koch's postulates, leaves of five A. japonica plants were wounded with a sterilized hypodermic needle and inoculated with 1 µL of a conidial suspension (106 conidia/mL). As control, 5 plants were inoculated with 1 µL of sterile water. After 7 days in a moist climate chamber at 26°C under a 16 h/8 h light/dark cycle, symptoms appeared on all inoculated leaves, while the control leaves remained healthy. The pathogen was reisolated from the inoculated leaves, and was confirmed to be C. boninense using the same morphological and molecular methods as before. C. boninense has been reported to infect many plant species, including Capsicum annuum, Rosa chinensis, and Eucalyptus robusta (Ding et al. 2021; Tozze et al. 2009; Zhang et al. 2018). Another Colletotrichum pathogen, C. fructicola, was reported to cause anthracnose on A. japonica in China (Gong et al. 2016). To our knowledge, this is the first report of A. japonica anthracnose disease caused by C. boninense in Guizhou province, China. Identifying this pathogen provides a foundation to prevent this complex disease and to reduce economic loss.

Gut bacteria reflect the adaptation of Diestrammena japanica (Orthoptera: Rhaphidophoridae) to the cave.

The gut microbiota is essential for the nutrition, growth, and adaptation of the host. Diestrammena japanica, a scavenger that provides energy to the cave ecosystem, is a keystone species in the karst cave in China. It inhabits every region of the cave, regardless of the amount of light. However, its morphology is dependent on the intensity of light. Whether the gut bacteria reflect its adaptation to the cave environment remains unknown. In this research, D. japanica was collected from the light region, weak light region, and dark region of three karst caves. The gut bacterial features of these individuals, including composition, diversity, potential metabolism function, and the co-occurrence network of their gut microbiota, were investigated based on 16S rRNA gene deep sequencing assay. The residues of amino acids in the ingluvies were also evaluated. In addition, we explored the contribution of gut bacteria to the cave adaptation of D. japanica from three various light zones. Findings showed that gut bacteria were made up of 245 operational taxonomic units (OTUs) from nine phyla, with Firmicutes being the most common phylum. Although the composition and diversity of the gut bacterial community of D. japanica were not significantly different among the three light regions, bacterial groups may serve different functions for D. japanica in differing light strengths. D. japanica has a lower rate of metabolism in cave habitats than in light regions. We infer that the majority of gut bacteria are likely engaged in nutrition and supplied D. japanica with essential amino acids. In addition, gut bacteria may play a role in adapting D. japanica's body size. Unveiling the features of the gut bacterial community of D. japanica would shed light on exploring the roles of gut bacteria in adapting hosts to karst cave environments.

Differential Function of Endogenous and Exogenous Abscisic Acid during Bacterial Pattern-Induced Production of Reactive Oxygen Species in Arabidopsis.

Abscisic acid (ABA) plays important roles in positively or negatively regulating plant disease resistance to pathogens. Here, we reassess the role of endogenous and exogenous ABA by using: 35S::ABA2, a previously reported transgenic Arabidopsis line with increased endogenous ABA levels; aba2-1, a previously reported ABA2 mutant with reduced endogenous ABA levels; and exogenous application of ABA. We found that bacterial susceptibility promoted by exogenous ABA was suppressed in 35S::ABA2 plants. The 35S::ABA2 and aba2-1 plants displayed elevated and reduced levels, respectively, of bacterial flagellin peptide (flg22)-induced H2O2. Surprisingly, ABA pre-treatment reduced flg22-induced H2O2 generation. Exogenous, but not endogenous ABA, increased catalase activity. Loss of nicotinamide adenine dinucleotide phosphate oxidase genes, RBOHD and RBOHF, restored exogenous ABA-promoted bacterial susceptibility of 35S::ABA2 transgenic plants. In addition, endogenous and exogenous ABA had similar effects on callose deposition and salicylic acid (SA) signaling. These results reveal an underlying difference between endogenous and exogenous ABA in regulating plant defense responses. Given that some plant pathogens are able to synthesize ABA and affect endogenous ABA levels in plants, our results highlight the importance of reactive oxygen species in the dual function of ABA during plant-pathogen interactions.

A type III effector XopLXcc8004 is vital for Xanthomonas campestris pathovar campestris to regulate plant immunity.

Xanthomonas campestris pv. campestris (Xcc) secretes a suite of effectors into host plants via the type III secretion system (T3SS), modulating plant immunity defenses. Strain Xcc8004 causes black rot in brassica plants, including Arabidopsis thaliana, making it a classical model for the study of Xanthomonas pathogenesis. XopLXcc8004 was defined as a T3SS effector (T3SE) since its homologues XopLXcv85-10 from Xanthomonas campestris pv. vesicatoria (Xcv85-10) contribute to virulence in host plants. Except for its virulence on Chinese radish plants, little was previously known about the regulation and function of XopLXcc8004. Here, we tested the role of XopLXcc8004 in the pathogenicity of Xcc8004 on different host plants including Arabidopsis. We found that it was required for full virulence of Xcc8004 in Chinese cabbage. XopLXcc8004 promoted bacterial infection in Arabidopsis and suppressed bacterial flagellin (flg22)-induced FRK1 transcription, reactive oxygen species (ROS) burst, callose deposition, and pathogenesis-related marker gene expression, but it did not affect mitogen-activated protein kinases (MAPKs) cascade. Early and prolonged expression of XopLXcc8004 affected Arabidopsis growth and development. We demonstrated that XopLXcc8004 is a virulence factor and interferes with innate immunity of Arabidopsis by suppressing pathogen-associated molecular pattern-triggered immunity (PTI) signaling, independent of MAPKs.

The Xanthomonas campestris effector protein XopDXcc8004 triggers plant disease tolerance by targeting DELLA proteins.

Plants protect themselves from the harmful effects of pathogens by resistance and tolerance. Disease resistance, which eliminates pathogens, can be modulated by bacterial type III effectors. Little is known about whether disease tolerance, which sustains host fitness with a given pathogen burden, is regulated by effectors. Here, we examined the effects of the Xanthomonas effector protein XopDXcc8004 on plant disease defenses by constructing knockout and complemented Xanthomonas strains, and performing inoculation studies in radish (Raphanus sativus L. var. radiculus XiaoJinZhong) and Arabidopsis plants. XopDXcc8004 suppresses disease symptoms without changing bacterial titers in infected leaves. In Arabidopsis, XopDXcc8004 delays the hormone gibberellin (GA)-mediated degradation of RGA (repressor of ga1-3), one of five DELLA proteins that repress GA signaling and promote plant tolerance under biotic and abiotic stresses. The ERF-associated amphiphilic repression (EAR) motif-containing region of XopDXcc8004 interacts with the DELLA domain of RGA and might interfere with the GA-induced binding of GID1, a GA receptor, to RGA. The EAR motif was found to be present in a number of plant transcriptional regulators. Thus, our data suggest that bacterial pathogens might have evolved effectors, which probably mimic host components, to initiate disease tolerance and enhance their survival.

[Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different Newcastle-disease isolates].

Thirteen prevailed Newcastle-disease viruses (NDV) isolated in China during 2001-2004 were purified by chick embryo fibroblast (CEF) plaque assay and characterized pathotypically and genotypically. The biological tests showed that these viruses were highly virulent. Sequence analysis based on the variable region (nucleotide 47-420) of the F gene indicated that of the 13 NDV isolates 2 belonged to genotype II, 2 to genotype IX and 9 to genotype VII. Isolates with genotype VII shared 94.6%-99.3% nucleotide (nt) homology with the F gene, whereas for genotype VII and La Sota was only 82.7%-84.1%. In addition, these NDV isolates all shared 95.2%-100% nt homology with the hemagglutinin-neuraminidase (HN) gene, whereas only 79.1%-84.3% compared these viruses with La Sota. The cross neutralization assays were done using positive serums in specific pathogen free (SPF) chicken embryos respectively. Correlation of the neutralization index in chicken embryo with the homologies of F and HN gene of different NDV isolates were analyzed by SPSS8.0 software. The result showed that the neutralization index was closely correlated with nt sequence (P < 0.01, r = 0.35) or deduced amino acid sequence (P < 0.01, r = 0.34) of the HN gene, whereas weekly correlated (P < 0.05, r = 0.20 or 0.19) with the F gene, and non-correlated with 374 nt segment. This implied that the genetic mutations of HN resulted in antigenic variations of these viruses and the search for new vaccines would be necessary.

Pathotypical characterization and molecular epidemiology of Newcastle disease virus isolates from different hosts in China from 1996 to 2005.

Thirty Newcastle disease virus (NDV) strains isolated from outbreaks in China during 1996 to 2005 were characterized pathotypically and genotypically. All strains except one were velogenic. An analysis of the variable region (nucleotides 47 to 420) of the F gene indicated that 6 isolates belonged to genotype II, 3 to genotype III, 1 (isolated from a pigeon) to genotype VI, and 20 to genotype VII. Isolates belonging to genotype VII were further divided into five subtypes, VIIa, VIIb, VIIc, VIId, and VIIe, and subtype VIId was made up of VIId1 to VIId5. These results showed that genotype VII isolates might have been the most prevalent in China during the past two decades. Genotype VII isolates shared high homology, but the homology was less than that between genotype VII viruses and the vaccine virus LaSota. Among these NDV isolates, 25 isolates had the velogenic motif (112)R/K-R-Q-K/R-R-F(117) that is consistent with results of the biological tests. However, four of five LaSota-type isolates that contained the lentogenic motif (112)G-R-Q-G-R-L(117) were velogenic, except SY/03, in the view of the biological test. The majority of genotype VII isolates had lost one or two N-glycosylation sites. Finally, a cross-protection experiment in which specific-pathogen-free chickens vaccinated with LaSota were challenged by six NDV isolates showed that more than three isolates were antigenic variants that could be responsible for recent outbreaks of Newcastle disease.