Genome Analysis of Pseudomonas viciae G166 Conferring Antifungal Activity in Grapevine.

Grapevine (Vitis vinifera) is one of the major economic fruit crops but suffers many diseases, causing damage to the quality of grapes. Strain G166 was isolated from the rhizosphere of grapevine and was found to exhibited broad-spectrum antagonistic activities against fungal pathogens on grapes in vitro, such as Coniella diplodiella, Botrytis cinerea, and Colletotrichum gloeosporioides. Whole-genome sequencing revealed that G166 contained a 6,613,582 bp circular chromosome with 5749 predicted coding DNA sequences and an average GC content of 60.57%. TYGS analysis revealed that G166 belongs to Pseudomonas viciae. Phenotype analysis indicated that P. viciae G166 remarkably reduced the severity of grape white rot disease in the grapevine. After inoculation with C. diplodiella, more H2O2 and MDA accumulated in the leaves and resulted in decreases in the Pn and chlorophyll content. Conversely, G166-treated grapevine displayed less oxidative damage with lower H2O2 levels and MDA contents under the pathogen treatments. Subsequently, G166-treated grapevine could sustain a normal Pn and chlorophyll content. Moreover, the application of P. viciae G166 inhibited the growth of mycelia on detached leaves and berries, while more disease symptoms occurred in non-bacterized leaves and berries. Therefore, P. viciae G166 served as a powerful bioagent against grape white rot disease. Using antiSMASH prediction and genome comparisons, a relationship between non-ribosomal peptide synthase clusters and antifungal activity was found in the genome of P. viciae G166. Taken together, P. viciae G166 shows promising antifungal potential to improve fruit quality and yield in ecological agriculture.

A Simulation Study to Reveal the Epidemiology and Aerosol Transmission Characteristics of Botrytis cinerea in Grape Greenhouses.

Most previously studies had considered that plant fungal disease spread widely and quickly by airborne fungi spore. However, little is known about the release dynamics, aerodynamic diameter, and pathogenicity threshold of fungi spore in air of the greenhouse environment. Grape gray mold is caused by Botrytis cinerea; the disease spreads in greenhouses by spores in the air and the spore attaches to the leaf and infects plant through the orifice. In this study, 120 µmol/L propidium monoazide (PMA) were suitable for treatment and quantitation viable spore by quantitative real-time PCR, with a limit detection of 8 spores/mL in spore suspension. In total, 93 strains of B. cinerea with high pathogenicity were isolated and identified from the air samples of grapevines greenhouses by a portable sampler. The particle size of B. cinerea aerosol ranged predominately from 0.65-3.3 µm, accounting for 71.77% of the total amount. The B. cinerea spore aerosols were infective to healthy grape plants, with the lowest concentration that could cause disease being 42 spores/m3. Botrytis cinerea spores collected form six greenhouse in Shandong Province were quantified by PMA-qPCR, with a higher concentration (1182.89 spores/m3) in May and June and a lower concentration in July and August (6.30 spores/m3). This study suggested that spore dispersal in aerosol is an important route for the epidemiology of plant fungal disease, and these data will contribute to the development of new strategies for the effective alleviation and control of plant diseases.

First report of Coniella vitis causing white rot on Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) in China.

Virginia creeper (Parthenocissus quinquefolia [L.] Planch.) belongs to the genus of Parthenocissus and Vitaceae family, which is very common in vineyards and where wild grape occurs (Bergh et al., 2011). In September of 2021, a severe white rot disease was observed on Virginia creeper around the vineyard of wine grapevine (Cabernet Sauvignon) located in Penglai city (37º 75'38" N, 120º 84'28" E), Shandong province of China. The disease incidence was about 75%, and infected leaf of Virginia creeper exhibited irregular necrotic lesion with brown center, and most lesion occurred on leaf margin, black pycnidia were also observed on the infected leaf at the late stage of infection. To determine the causal agent, symptomatic leaves with typical lesions were cut into small pieces (5 mm × 3 mm), surface sterilized with 75% ethanol for 1 min, followed by three times rinsed in sterile water. Leaf sections were plated onto potato dextrose agar (PDA) medium and incubated at 28°C for 3 days. Totally, five isolates (referred to as JD01, JD07, JD09, JD12 and JD16) were collected and transferred on to fresh PDA medium for incubation at 28°C. Seven days later, colonies on PDA plates had crenulated edges with concentric rings, the upper surface of colonies was mostly flat and white with many pycnidia. The conidia were hyaline at immature and became brown later, spherical or ellipsoid, aseptate, and 7.92 ± 1.20 µm × 5.18 ± 0.61 µm (n=50), length : width ratio is nearly 2. Morphologically, the isolates were identified as Coniella vitis (Chethana et al., 2017). Further to confirm the fungal species, the internal transcribed spacer region (ITS) of the ribosomal RNA gene, large subunit rRNA gene (LSU) and the translation elongation factor 1-alpaha gene (TEF1-α) were amplified using primers ITS1/ ITS4, LR7/ LROR, and TEF1- 728F/ TEF1- 986R (Chethana et al., 2017; Raudabaugh et al., 2018). The amplification products were sequenced and deposited in GenBank database. The sequences were compared to type sequences in GenBank. The results showed that ITS (GenBank accession numbers ON329769, ON329770, ON329771, ON329772 and ON329773), LSU (ON358423，ON358424, ON358425, ON358426 and ON358427) and TEF (ON297671, ON229071, ON229072, ON229073 and ON297672) sequences of the five isolates were 99.66%, 96.90% and 98.79% identical with the sequences data from C. vitis isolates in GeneBank (MFLUCC 18-0093, JZB3700020 and MFLUCC 18-0093, respectively). Furthermore, concatenated sequences of the three genes (ITS, LSU and TEF) were used to conduct a phylogenetic tree using maximum likehood MEGA-X (Raudabaugh et al., 2018). The phylogenetic analysis showed that the five isolates (JD01, JD07, JD09, JD12 and JD16) belong to C. vitis clade among the 41strains of Coniella spp. In the pathogenicity tests, detached leaves of Virginia creeper (1-year-old) were inoculated with mycelia plugs (5 mm diameter) (form 3-day-old of isolate JD07 culture), and control were inoculated with PDA plugs (5 mm diameter). Virginia creeper live plants (1-year-old) were inoculated with conidial suspension (2.5×106 spores/ml) of the isolate JD07 of one week old, and control plants were inoculated with sterile water. All treated Virginia creeper plants (detached leaves) were placed in a greenhouse maintained at 28°C and 95% relative humidity. Virginia creeper plants (detached leaves) inoculated with the conidial suspension (fungal mycelia) had brown lesion on leaves, the disease symptoms were similar to those observed in field. No such symptoms were observed on control plants (detached leaves). The pathogen was reisolated from inoculated Virginia creeper plants and re-identified, thus fulfilling Koch's postulates. C. vitis had been reported to cause grape white rot in China (Chethana et al., 2017). Virginia creeper, as an excellent host of C. vitis, will increase the transmission risk of the pathogens. To our knowledge, this is the first report of C. vitis causing white rot on Virginia creeper, and this finding will provide useful information for developing effective control strategies for white rot disease.

Comparative genomic and functional analyses of Paenibacillus peoriae ZBSF16 with biocontrol potential against grapevine diseases, provide insights into its genes related to plant growth-promoting and biocontrol mechanisms.

Paenibacillus peoriae is a plant growth-promoting rhizobacteria (PGPR) widely distributed in various environments. P. peoriae ZBFS16 was isolated from the wheat rhizosphere and significantly suppressed grape white rot disease caused by Coniella vitis. Here, we present the complete genome sequence of P. peoriae ZBFS16, which consists of a 5.83 Mb circular chromosome with an average G + C content of 45.62%. Phylogenetic analyses showed that ZBFS16 belongs to the genus P. peoriae and was similar to P. peoriae ZF390, P. peoriae HS311 and P. peoriae HJ-2. Comparative analysis with three closely related sequenced strains of P. peoriae identified the conservation of genes involved in indole-3-acetic acid production, phosphate solubilization, nitrogen fixation, biofilm formation, flagella and chemotaxis, quorum-sensing systems, two-component systems, antimicrobial substances and resistance inducers. Meanwhile, in vitro experiments were also performed to confirm these functions. In addition, the strong colonization ability of P. peoriae ZBFS16 was observed in soil, which provides it with great potential for use in agriculture as a PGPR. This study will be helpful for further studies of P. peoriae on the mechanisms of plant growth promotion and biocontrol.

Suppression of Grape White Rot Caused by Coniella vitis Using the Potential Biocontrol Agent Bacillus velezensis GSBZ09.

Grape white rot caused by Coniella vitis is prevalent in almost all grapevines worldwide and results in a yield loss of 10-20% annually. Bacillus velezensis is a reputable plant growth-promoting bacterial. Strain GSBZ09 was isolated from grapevine cv. Red Globe (Vitis vinifera) and identified as B. velezensis according to morphological, physiological, biochemical characteristics and a multilocus gene sequence analysis (MLSA) based on six housekeeping genes (16S rRNA, gyrB, rpoD, atpD, rho and pgk). B. velezensis GSBZ09 was screened for antifungal activity against C. vitis under in vitro and in vivo conditions. GSBZ09 presented broad spectrum antifungal activity and produced many extracellular enzymes that remarkably inhibited the mycelial growth and spore germination of C. vitis. Furthermore, GSBZ09 had a high capacity for indole-3-acetic acid (IAA) production, siderophore production, and mineral phosphate solubilization. Pot experiments showed that the application of GSBZ09 significantly decreased the disease index of the grape white rot, directly promoted the growth of grapes, and upregulated defense-related enzymes. Overall, the features of B. velezensis GSBZ09 make it a potential strain for application as a biological control agent against C. vitis.

Diversity and spoilage potential of microbial communities associated with grape sour rot in eastern coastal areas of China.

As a polymicrobial disease, sour rot decreases grape berry yield and wine quality. The diversity of microbial communities in sour rot-affected grapes depends on the cultivation site, but the microbes responsible for this disease in eastern coastal China, has not been reported. To identify the microbes that cause sour grape rot in this important grape-producing region, the diversity and abundance of bacteria and fungi were assessed by metagenomic analysis and cultivation-dependent techniques. A total of 15 bacteria and 10 fungi were isolated from sour rot-affected grapes. High-throughput sequencing of PCR-amplicons generated from diseased grapes revealed 1343 OTUs of bacteria and 1038 OTUs of fungi. Proteobacteria and Firmicutes were dominant phyla among the 19 bacterial phyla identified. Ascomycota was the dominant fungal phylum and the fungi Issatchenkia terricola, Colletotrichum viniferum, Hanseniaspora vineae, Saprochaete gigas, and Candida diversa represented the vast majority ofmicrobial species associated with sour rot-affected grapes. An in vitro spoilage assay confirmed that four of the isolated bacteria strains (two Cronobacter species, Serratia marcescens and Lysinibacillus fusiformis) and five of the isolated fungi strains (three Aspergillus species, Alternaria tenuissima, and Fusarium proliferatum) spoiled grapes. These microorganisms, which appear responsible for spoiling grapes in eastern China, appear closely related to microbes that cause this plant disease around the world.