Comparison of Copper-Tolerance Genes between Different Groups of Acidovorax citrulli.

Acidovorax citrulli populations exhibit genetic and phenotypic variations, particularly in terms of copper tolerance. Group I strains of A. citrulli generally exhibit higher copper tolerance compared to group II strains. This study aims to identify genes involved in copper tolerance to better understand the differences in copper tolerance between group I and group II strains. Representative strains pslb65 (group I) and pslbtw14 (group II) were selected for comparison. Deletion mutants of putative copper-tolerance genes and their corresponding complementary strains were constructed. The copper tolerance of each strain was evaluated using the minimum inhibitory concentration method. The results showed that the copA, copZ, cueR, and cueO genes played major roles in copper tolerance in A. citrulli, while cusC-like, cusA-like, and cusB-like genes had minor effects. The different expression levels of copper-tolerance-related genes in pslb65 and pslbtw14 under copper stress indicated that they had different mechanisms for coping with copper stress. Overall, this study provides insights into the mechanisms of copper tolerance in A. citrulli and highlights the importance of specific genes in copper tolerance.

PlantPAD: a platform for large-scale image phenomics analysis of disease in plant science.

Plant disease, a huge burden, can cause yield loss of up to 100% and thus reduce food security. Actually, smart diagnosing diseases with plant phenomics is crucial for recovering the most yield loss, which usually requires sufficient image information. Hence, phenomics is being pursued as an independent discipline to enable the development of high-throughput phenotyping for plant disease. However, we often face challenges in sharing large-scale image data due to incompatibilities in formats and descriptions provided by different communities, limiting multidisciplinary research exploration. To this end, we build a Plant Phenomics Analysis of Disease (PlantPAD) platform with large-scale information on disease. Our platform contains 421 314 images, 63 crops and 310 diseases. Compared to other databases, PlantPAD has extensive, well-annotated image data and in-depth disease information, and offers pre-trained deep-learning models for accurate plant disease diagnosis. PlantPAD supports various valuable applications across multiple disciplines, including intelligent disease diagnosis, disease education and efficient disease detection and control. Through three applications of PlantPAD, we show the easy-to-use and convenient functions. PlantPAD is mainly oriented towards biologists, computer scientists, plant pathologists, farm managers and pesticide scientists, which may easily explore multidisciplinary research to fight against plant diseases. PlantPAD is freely available at http://plantpad.samlab.cn.

First Report of Bayberry Leaf Blight Caused by Pestalotiopsis kenyana in Zhejiang Province, China.

Weizhi Xun and Changwang Wu contributed equally to this work In October 2020, bayberry (Myrica rubra (Lour.) S. et Zucc.) leaves that beginning to wither were collected in Wencheng County (N27°50', E120°03'). In the county, 4,120 ha of bayberry were planted, of which 58% were affected by the disease, and the severity of leaf disease per plant was 5 to25%. Bayberry leaves leaves were intensely green at first, then gradually turned yellow and brown,and completely withered. The leaves did not fall off at the beginning of the symptoms, but did fall after 1 to 2 months. To identify the pathogen, 50 diseased leaves with typical symptoms were collected from 10 diseased trees. Leaves with necrotic-tissue were firstly washed with sterilized water, and then tissue at the disease-/ healthy-tissuejunction removed with sterile surgical scissors. The tissues were soaked in 75% ethanol for 30 s, followed by 5% sodium hypochlorite solution for 3 to 4 min, rinsed with sterilized water 4 times, and placed on sterilized filter paper. The tissue was placed on PDA medium and cultured in an incubator at 25℃ (Nouri et al. 2019). After the colonies grew around the tissue, mycelia with the same morphology was selected and placed on fresh PDA. A pure culture of the pathogen was obtained after repeating the last process several times. The isolatedcolonies were white, with a round edge and a light-yellow back. Conidia were straight or slightly curved, with 3 to 4 septations. The internal transcribed spacer (ITS) regin translation elongation factor 1-α gene (TEF1-α), and beta-tubulin gene (ß-TUB)(Chaiwan et al. 2020; Li et al. 2021; Chen et al. 2020; Chen et al. 2018) of the two strains were amplified and sequenced, and the sequences were uploaded to Gen bank (GenBank accession number.ACCC 35162: ITS OP891011, TEF1-α OP903533, ß-TUB OP903531; ACCC 35163: ITS OP891012, ß-TUB OP903534, TEF1-α OP903532). BLAST alignment indicated that the ITS sequence of strain ACCC 35162 had 100% identity with NR_147549.1, the TEF sequence had 100% identity with MT552449.1, and the TUB sequence had 99.87% identity with KX895323.1; the ITS sequence of strain ACCC 35163 had 100% identity with NR_147549.1, the TEF sequence had 100% identity with MT552449.1, and the TUB sequence had 99.86% identity with KX895323.1. A Phylogenetic tree using maximum likelihood/rapid bootstrapping run on XSEDE based on the above three sequences inferred that the two strains were identical to P. kenyana (Miller et al. 2010). The strain was preserved in the Agricultural Culture Collection of China (Preservation numbers: ACCC 35162, ACCC 35163). Following Koch's rule, six healthy plants leaves were inoculated with conidial suspensions (106 conidia mL-1) and mycelial plugs (5 mm)，and then placed in an artificial climate chamber (25℃, 90% humidity, 16-h light), sterile PDA and sterile water were used as blank controls. The same treatment was applied to fresh bayberry leaves under laboratory conditions, and brown spots were observed after three days. There were no symptoms in the control group. The experimental symptoms were similar to those in the field. Using the previous method, the same fungus was reisolated from the diseased leaves and again identified as P. kenyana. As far as we know, this is the first report causing disease on P. kenyana infecting bayberry in China, this disease seriously affected the yield and quality of bayberry and caused economic losses to farmers.

ntrC Contributes to Nitrogen Utilization, Stress Tolerance, and Virulence in Acidovorax citrulli.

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, severely damages watermelon, melon, and other cucurbit crops worldwide. Nitrogen, one of the most important limiting elements in the environment, is necessary for the growth and reproduction of bacteria. As a nitrogen-regulating gene, ntrC plays an important role in maintaining bacterial nitrogen utilization and biological nitrogen fixation. However, the role of ntrC has not been determined for A. citrulli. In this study, we constructed a ntrC deletion mutant and a corresponding complementary strain in the background of the A. citrulli wild-type strain, Aac5. Through phenotype assays and qRT-PCR analysis, we investigated the role of ntrC in A. citrulli in nitrogen utilization, stress tolerance, and virulence against watermelon seedlings. Our results showed that the A. citrulli Aac5 ntrC deletion mutant lost the ability to utilize nitrate. The ntrC mutant strain also exhibited significantly decreased virulence, in vitro growth, in vivo colonization ability, swimming motility, and twitching motility. In contrast, it displayed significantly enhanced biofilm formation and tolerance to stress induced by oxygen, high salt, and copper ions. The qRT-PCR results showed that the nitrate utilization gene nasS; the Type III secretion system-related genes hrpE, hrpX, and hrcJ; and the pili-related gene pilA were significantly downregulated in the ntrC deletion mutant. The nitrate utilization gene nasT, and the flagellum-related genes flhD, flhC, fliA, and fliC were significantly upregulated in the ntrC deletion mutant. The expression levels of ntrC gene in the MMX-q and XVM2 media were significantly higher than in the KB medium. These results suggest that the ntrC gene plays a pivotal role in the nitrogen utilization, stress tolerance, and virulence of A. citrulli.

Identification of the Causal Agent of Bacterial Leaf Spot on Watermelon in China.

Watermelon diseases caused by pathogenic bacteria were endemic in Liaoning and Jilin Provinces from 2019 to 2020 in China, resulting in serious economic losses to the watermelon industry. This study characterized 56 strains isolated from symptomatic watermelon leaves collected from Liaoning and Jilin Provinces. Through morphological observation, 16S rRNA and gyrB sequence analysis, and BIOLOG profiles, the pathogen was identified as Pseudomonas syringae. In China, the watermelon disease caused by P. syringae was reported for the first time. The multilocus sequence analysis showed that the isolated strains belonged to three different clades within P. syringae phylogroup 2. Interestingly, most of them (79%) belonged to clade 2a, 14% were clade 2b, and 7% were clade 2d. This indicates that bacterial leaf spot outbreaks of watermelon in China were caused by multiple sources and mainly by P. syringae clade 2a.

[Cloning and expressing of a harpin-encoding gene from Pseudomonas syringae pv. glycinea].

UNLABELLED: METHODS, OBJECTIVE: We amplified the 1026 bp hrp (hypersensitive response and pathogenicity) gene from Pseudomonas syringae pv. glycinea isolate Psg12 genomic DNA by PCR technique, and then constructed expression vector pGEX-hrpZ(Psg12) with regular molecular cloning operation. The recombinant plasmid was transformed into BL21(DE3). Recombinant protein was induced by Isopropylthio-beta-D-Galacgoside (IPTG). RESULTS: The molecular mass of the fusion protein is 61kDa analyzed by SDS-PAGE. The protein, similar to the other known harpins, was heat-stable, which contained abundant glycine(G), but had no cysteine. Furthermore, this protein was sensitive to protease K and able to trigger hypersensitive response (HR) in common tobacco. The HR elicitation by the protein in tobacco was inhibited by eukayotic metabolic inhibitors, NH4 VO3 and LaCl3. The hrpZ gene showed 79% identity to hrpZ(Psg) which cloned from P. syringae pv. glycinea (Psg r0) in Japan and 79 - 99% identity to other hrpZ in GenBank. However, it did not show any sequence identity with those of other genus of gram-negative plant pathogenic bacteria. CONCLUSION: In summary, hrpZ(Psg12) was a novel gene that was cloned by us from P. syringae pv. glycinea, and this is the first report to express hrpZ(Psg12) gene in BL21.

Effect of cDNA fragments in different length derived from potato virus Y coat protein gene on the induction of RNA-mediated virus resistance.

We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3' end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the transgenic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3' end of PVY CP gene.