Bacterial leaf spot of tomato is a worldwide disease, which seriously affects the quality and yield of tomato because of its diverse routes of infestation, rapid spread and difficulty in control. To identify the pathogen of the disease, samples of tomato with leaf spots were collected and prepared for pathogen analysis. Five representative bacterial strains FQ21051401-FQ21051405 strains were isolated and purified from diseased tomato plants. Pathogenicity tests and re-isolation and re-identification of the bacteria were performed to confirm the isolate and fulfill the Koch′ postulates. Based on morphological and biochemical characteristics, phylogenetic analysis, and Koch′s postulates, the bacterial isolates were identified as Erwinia persicina. The aim of this study was to isolate and identify the causal agent of bacterial leaf spot disease of tomato, which will provide a better understanding of pathogenesis of the E. persicina and provided a scientific basis for its control.
Common bean（Phaseolus vulgaris）, also known as the Kidney bean, is widely grown in China for its fresh pod or edible dry seeds, which consumed as vegetable and cereal. In July 2021, severe outbreaks of common bean wilt were observed in several commercial fields in Inner Mongolia. A fungus was isolated from dark brown lesions on diseased stems and roots. The pathogenicity of the isolated fungus was confirmed from inoculation experiments. On the basis of morphological features and EF-1α and β-tubulin sequences, the pathogen was identified as Fusarium oxysporum. This is the first report of F. oxysporum causing Fusarium wilt on common bean in Inner Mongolia. As a destructive plant pathogen, F. oxysporum has a wide host range and is distributed all over China, control measures should be taken in advance.
薄壳山核桃（Carya illinoinensis （Wangenh.） K. Koch）又称美国山核桃，是绿化的优选树种，其果实又称碧根果是世界著名的干果之一。薄壳山核桃原产于美国和墨西哥北部，因其较高的经济价值已被引种到全球20多个国家和地区 [1-2] 。江苏是我国薄壳山核桃引种较早的省份之一 [3] ，江苏句容拥有万亩薄壳山核桃基地，发展势头良好。随着薄壳山核桃种植面积的扩大，病虫害问题突显，褐斑病在江苏句容相继发生，导致叶斑，果腐，落果，经济损失严重。从江苏句容两个薄壳山核桃种植区采集发病样品，进行病原菌的分离与鉴定，结果报道如下。
Verticillium dahliae is a worldwide phytopathogenic fungus which can infect more than 660 plant species, causing serious economic losses annually. Genomic analysis of V. dahlia revealed that different strains contained many strain-specific genes, however, the biological functions of most genes were not clear. In our study, by comparing the genomes of V. dahliae VdLs17 and JR2, a JR2 strain-specific gene Chr6g02370 was identified, which encodes alcohol dehydrogenase and was induced during infection. The enzymatic activity of ethanol dehydrogenase in the knockout mutant decreased significantly, but the utilization of carbon sources such as ethanol was not affected. In addition, the virulence of knockout mutants to tobacco and tomato was significantly reduced. The phenotypic analysis also showed that knockout of this gene reduced melanin synthesis ability, but did not affect mycelial growth and sporulation. In conclusion, our results expand the understanding of the biological functions of strain-specific genes in V. dahlia and provide genetic material for further functional genomics.
In order to understand the resistance risk of Alternaria alternata to mefentrifluconazole, four mutant strains with different resistance levels were obtained by chemical domestication in this study. The resistance risk of A. alternata was evaluated by detecting genetic stability, biological characteristics and cross resistance between mefentrifluconazole and other fungicides. The results showed that the high resistance mutant had high fitness and stable inheritance. Once it appears, it is at risk of becoming a dominant strain in the field. There was no positive cross resistance between the resistance of mefentrifluconazole resistant mutants to mefentrifluconazole and the resistance of other five fungicides, azoxystrobin, kersonxim-methyl, difenoconazole, pyraclostrobin, and hexaconazole. A. alternata can develop mefentrifluconazole-resistant mutants under the fungicide pressure, and there is a risk of fungicide resis-tance. The results of this study provide a scientific basis for the reasonable application of mefentrifluconazole for the control of ginseng Alternaria leaf blight.
From 2019 to 2021, a new leaf spot disease was detected on Salvia miltiorrhiza Bge. in Henan province. The leaf spot disease could be found in all 12 investigated fields, and the rate of diseased plants was 20% to 70%. The disease spot was round or oval, gray white in the center, and appeared perforation at later stages which eventually led to leaf wilt by meeting together with multiple spots. The pathogen was isolated and purified by conventional tissue isolation, and was verified according to Koch′s rule. Based on morphological observation and rDNA-ITS, EF-1α and β-tubulin gene sequence analysis, isolate DS1 was identified as Corynespora cassiicola, the causal agent of the new leaf spot disease. This is the first report of leaf spot disease on S. miltiorrhiza caused by C. cassiicola in China, which will provide theoretical basis and technical support for the effective control of the disease in the planting and production of S. miltiorrhiza in the future.
In order to study the systemic infection of Verticillium dahliae in potato, we firstly established a Petri dish filter paper system by which the evaluation of dynamic roots infection in resistant/susceptible potato varieties was conducted with the GFP fluorescence labelled Verticillium dahliae strain. The results showed that after soaking for 2 hours in a conidia suspension of 10~7conidia·mL -1 , conidia could attach to the root hairs of resistant/susceptible potato roots, but a large amount of conidia was attached to the roots of susceptible varieties in which 65 conidia were on susceptible cultivar, while 23 conidia on resistant cultivar at average view of microscope. After 12 hours of inoculation, there was no significant difference in the germination rate of conidia on the roots surface of both resistant and susceptible varieties. Three days after inoculation, the mycelium could penetrate the root epidermis and enter the intercellular space of tissues. However, compared with the resistant varieties, the mycelium in the root system of the susceptible varieties grew faster and reached the inner vascular bundle of the root system at 7 dpi, and the green fluorescence signal was observed in the vascular bundle of resistant varieties only at 9 dpi. At 10 dpi, new conidia stalk and conidia formation could be seen in the roots of susceptible varieties, while no conidia formation in the resistant varieties. The amount of pathogen colonization in the aerial stems of susceptible potato varieties at 30 dpi was five times more than that of the resistant varieties. At the same time, the number of pathogens in the petioles of susceptible varieties was also significantly higher than that of resistant varieties.
A Ganoderma stem rot disease was found to infect Dimocarpus longana in 2021 in Binqiao town of Guangxi province. In order to clarify the pathogen of this disease, ten isolates with the same morphology were isolated from basidiocarps by using aconventional tissue separation method. LYJF001, a representative strain with excellent growth, was selected for pathogenicity test, morphological identification, sequence analysis of rDNA-ITS and SSU, and phylogenetic tree construction. The results showed that the pathogen causing the stem rot of D. longana was Ganoderma australe.
Fusarium wilt of bitter gourd is a soil-borne disease caused by Fusarium oxysporum f. sp. momordicae Sun ＆ Huang（FOM）, which seriously affects the production of bitter gourd. Clarifying the pathogenic mechanism of FOM has important guiding significance for the prevention and control of Fusarium wilt of bitter gourd, but the pathogenic mechanism of FOM is still unclear. In the previous analysis of the differentially expressed genes in the transcriptomes of the strong pathogenic strain SD-1 and the weak pathogenic strain SD-V（carrying mycoviruses） of FOM, it was found that the expression level of FoHAD-type II was significantly down-regulated in SD-V strain. Therefore, we speculated that this gene may be involved in the pathogenic process of FOM. In this study, based on the principle of homologous recombination, the fusion fragment of FoHAD-type Ⅱ was obtained by Split-Marker PCR, the gene knockout mutant was obtained by PEG-mediated protoplast transformation, and the complementary mutant was obtained by Agrobacterium transformation method. The biological characteristics and pathogenicity of the mutants were tested and analyzed. The results showed that the growth rate and spore production of the FoHAD-type Ⅱ gene knockout mutant on the PDA medium were not significantly different from the wild-type strain, and there were no significant differences in the morphology of hyphal tips and spores. However, the vegetative growth of the knockout mutant was defective, manifested as different colony morphology, reduced aerial hyphae, significantly reduced virulence, and increased sensitivity to osmotic stress. The pathogenicity of the complemented mutant was consistent with that of the wild-type strain. Therefore, these findings suggested that FoHAD-type Ⅱ is not involved in the growth and development of FOM, but plays an important role in the pathogenic process of FOM, which may be related to the decline of pathogenicity of FOM caused by mycovirus. The results of this study provide a basis for the subsequent exploration of the pathogenic mechanism of FOM and a reference for the study of the hypovirulence mechanism of mycoviruses.
Rapid development and efficient mating are the basis of population expansion and disease epidemic of B. xylophilus. Bioinformatics analysis, in situ hybridization and RNA interference were used to investigate the potential role of Bxy-egl-30 in the growth and development of B. xylophilus. The cloned coding region of the Bxy-egl-30 gene has a length of 1 128 bp, encoding 375 amino acids, and the phylogenetic tree analysis showed that Bxy-egl-30 belonged to the EGL-30 protein subunit family. The results of in situ hybridization showed that the expression of Bxy-egl-30 was specific in different developmental stages of B. xylophilus, and it was widely expressed in the gonads, gut and nearby body wall cells, during embryonic and larval stages, the gene is expressed in the vulva muscle of females and the gonad of males. The results of RT-qPCR showed that the expression level of Bxy-egl-30 reached the highest level at the second larval stage, followed by the embryonic stage, and in the third instar stage, the fourth instar stage, and the adult stage, the expression level of Bxy-egl-30 decreased in turn. Knockdown of Bxy-egl-30 decreased the hatching rate of pinewood nematode embryos by 10.88%, and the activity of the second and third instar larvae decreased obviously, which the head swing frequency decreased about 4 times per minute. The results of the inoculation experiment showed that the pathogenicity of nematodes was weakened by Bxy-egl-30 RNAi and the death time of trees was slowed down. The death time of pine trees in the interference group was 9 days later than that in the control group. This study investigated the spatiotemporal dynamic expression patterns and biological functions of the Bxy-egl-30 gene at different deve-lopmental stages of B. xylophilus, and revealed the important role of the gene in the movement and development of B. xylophilus.
The role of miRNAs in regulating resistance to Sclerotinia sclerotiorum in oilseed rape（Brassica napus） is largely unknown. In this study, functions and mechanisms of miR6030 in resistance to S. sclerotiorum were analyzed. The results showed that miR6030 was expressed differentially in various tissues, higher in roots and stems while lowest in flowers. Silencing and transient over-expression of miR6030 in oilseed rape plants demonstrated that miR6030 negatively regulates resistance to S. sclerotiorum. The degradome analysis and GUS staining assay revealed that miR6030 targets BnaCnng64090D and BnaA09g10520D, which encode CC-NBS-LRR（CNL） proteins and positively regulate resistance to S. sclerotiorum. All the results indicated that miR6030 negatively regulates resistance to S. sclerotiorum via cleaving transcripts of the two CNL protein-encoding genes BnaCnng64090D and BnaA09g10520D. This study provides some insights into functions of miR6030 and molecular mechanisms underlying the resistance to S. sclerotiorum and new strategy for molecular breeding for the resistance of Brassica napus to S. sclerotiorum.
As one of the largest transcription factor families in plants, MYB transcription factors family play important roles in regulating plant growth and development and resisting to stress. However, the role of OsMYBs in rice antiviral pathway is still unclear. In order to explore the roles of OsMYBs family in rice antiviral pathway, we first analyzed their structure and characteristics, as well as the expression profiles of single-stranded RNA virus rice grassy stunt virus（RGSV） and double-stranded RNA virus rice ragged stunt virus（RRSV）-infected rice published by Mohammed et al in 2015, and found that 51 of the OsMYBs family genes responded to RGSV and 50 of them responded to RRSV. Further analysis revealed that 26 OsMYBs responded to both RGSV and RRSV, and 10 of them with relatively high expression and multiple differences were selected for real-time fluorescence quantitative PCR（qRT-PCR） verification. Our results showed that the expression of OsMYB30, LOC＿Os02g42850, LOC＿Os02g47744, LOC＿Os05g37050, LOC＿Os06g43090 and LOC＿Os08g33150 was down-regulated and the expression of LOC＿Os05g38460 was up-regulated upon RGSV and RRSV infection, respectively. Moreover, the expression of LOC＿Os05g51160 and LOC＿Os09g26170 was up-regulated after RGSV infection while down-regulated after RRSV infection; Additionally, the expression of LOC＿Os08g05520 was down-regulated after RGSV infection while up-regulated after RRSV infection. Seven out of these ten OsMYBs were identical in expression variation trend after infection of these two viruses, suggesting that these OsMYBs may have a broad-spectrum response to rice virus infection, which lays a foundation for future research on the function of OsMYBs during rice-virus interactions.
In order to detect the sensitivity of Fusarium pseudograminearum to epoxiconazole in Henan Pro-vince, 100 isolates were collected from 16 counties in 2019 and their sensitivity to epoxiconazole(EC 50 ) were conducted by mycelial growth rate method. The results indicated that the EC 50 of the tested isolates ranged from 0.002 4 to 0.505 4 μg·mL -1 , and the average value was（0.109 3±0.095 7） μg·mL -1 . The sensitivity frequency distribution showed that EC 50 values of the 89 tested isolates were lied in a main peak; the average value of these isolates（0.082 1±0.046 8） μg·mL -1 could be used as the sensitivity baseline of F. pseudograminearum to epoxiconazole. The results of variance analysis showed that there was little difference in the sensitivity of isolates from different counties to epoxiconazole with average EC 50 ranged from 0.050 8 to 0.296 8 μg·mL -1 . However, the sensitivities were varied for the isolates from the same county, and the highest EC 50 value was 210.58 times higher than that of the lowest isolates in Yucheng from Shangqiu. The results of cluster analysis showed that there was no obvious correlation between the sensitivity and the geographical origin of the isolates. There was no significant correlation between the toxicity of epoxiconazole and pyraclostrobin, fluoxonil, difenoconazole, carbendazim, phenamacril, tebuconazole. Seed dressing with epoxiconazole hindered wheat growth and inhibited root length, plant height and fresh weight. Control efficacy in greenhouse test illustrated that spraying 12.5% epoxiconazole suspension with active ingredient at 150 μL·mL -1 had the highest control efficacy of 60.18%. In vitro test, 12.5% epoxiconazole suspension not only had the highest control efficacy（79.78%） when the active ingredient at 150 μL·mL -1 , but had no effect on the growth of wheat when applied. The results of this study provided a theoretical basis for the application of epoxiconazole to control wheat stem rot and a basis for monitoring the sensitivity of the pathogen to epoxiconazole.
In order to clarify the occurrence and distribution of garlic virus in Shandong Province, 382 garlic samples were collected in 2019. PCR was used for virus detection with specific primers. Eleven viruses were detected: leek yellow stripe virus（LYSV）, onion yellow dwarf virus（OYDV）, garlic common latent virus（GCLV）, shallot latent virus（SLV）, garlic virus A（GarV-A）, garlic virus B（GarV-B）, garlic virus D（GarV-D）, garlic virus E（GarV-E）, garlic virus X（GarV-X）, milk vetch dwarf virus（MDV）and tobacco mosaic virus（TMV）. The co-infection rate of the viruses was 44.24%, and there was co-infection of six kinds of viruses at most. Phylogenetic trees were constructed with CP of LYSV and OYDV. LYSV isolates were closely related to the Chinese, Turkish and Mexican isolates. OYDV isolates were closely related to the Chinese, Indian and Polish isolates.
Endocytosis plays critical roles in cellular processes of nutrient uptake and signal transduction. Syp1 and Ede1 are early proteins in clathrin-mediated endocytosis. Fusarium head blight mainly caused by Fusarium graminearum seriously affects the yield and quality of wheat. F. graminearum contains one FgSYP1 that is highly homologous to Saccharomyces cerevisiae SYP1. Yeast two-hybrid showed that FgSyp1 interacted with F. graminearum FgEde1. The ΔSyp1 mutant and the ΔΔSyp1/Ede1 double mutant were obtained by homologous recombination, and the function of FgSyp1 was determined by experiments including hyphal growth, conidiation and pathogenicity. The results showed that FgSyp1 played an important role in hyphal growth, conidiation and pathogenicity of F. graminearum, and that the ΔΔSyp1/Ede1 mutant exhibited obvious defects in asexual and sexual reproduction compared with the ΔSyp1 mutant.
The WRKY transcription factors are a class of plant-specific transcription factor family. Studies have shown that many genes in the WRKY group III family are involved in defense response of pathogenic bacteria and other biotic stress. To study potential function of turnip WRKY group Ⅲ family genes in clubroot disease resistance, 21 WRKY genes were identified from the turnip genome using bioinformatics analysis and were distributed on the nine chromosomes. These WRKY proteins contain 258-916 amino acid residues with motif quantity ranging from 0-8, and most of them contain a motif structure with the sequence 4-5-7/（8-2）-1-3. Ana-lysis of cis-acting elements in promoter regions of WRKY group Ⅲ family gene found that there were a large number of light, hormone and stress responsive elements. Among hormone-responsive elements, Me-JA had the largest number. qPCR analysis showed that WRKY group Ⅲ family genes could respond to the induction of Plasmodiophora brassicae. Among them, BrWRKY46-2 and BrWRKY70-2 showed opposite patterns of up-and down-regulated expression in resistant and susceptible materials, respectively. The expression levels of BrWRKY46-2 and three BrWRKY70s were significantly down-regulated after exogenous SA induction, and were significantly down-regulated after MeJA induction. Subcellular localization showed that two genes, BrWRKY46-2 and BrWRKY70-2, mainly located in the nucleus. The WRKY group Ⅲ family genes in turnip were widely involved in the response process under P. brassicae stress. BrWRKY46-2 and BrWRKY70-2 genes were closely related to the resistance and susceptibility of turnip varieties and were induced by SA and JA. The above results imply that WRKY transcription factors have regulatory effects on clubroot resistance, and can be used as candidate genes for further needed on disease resistance and functional analysis.
As a famous medicinal plant, the growth and quality of Curcuma wenyujin can be affected by leaf sheath rot disease, which severely occurs in Dongshan plantations, Haikou city, Hainan province of China in 2017. Based on morphological traits and phylogenetic analyses of multi-loci（ITS, Cox I and Cox II）, the pathogen isolated from the diseased plants was identified as Pythium myriotylum. The results provide a theoretical basis for the field control of leaf sheath rot disease on C. wenyujin.
Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici（Pst）, seriously harms wheat production in China. Calmodulin-binding transcription activator（CAMTA） is the most vital one regulated by Ca 2+ /CaM in plants playing a significant role to biological stress. In this study, four wheat CAMTA gene family members down-regulated after Pst treatment were screened out from transcriptome database of the wheat-Pst interaction, and their functions in the interaction between wheat and stripe rust were preliminarily analyzed following real-time quantitative PCR（qRT-PCR） and virus-mediated gene silencing（VIGS） technologies. The results showed that in the compatible interaction, TaCAMTA1-A, TaCAMTA1-B, and TaCAMTA3-D were significantly down-regulated at different time points, while TaCAMTA2-B was obviously up-regulated at 12 h. VIGS-based knockdown of TaCAMTA2-B in wheat leaves was significantly reduced the number of necrotic spots and spores on the leaves, indicating that the disease resistance was enhanced. Therefore, the wheat TaCAMTA2-B gene was cloned, and tobacco subcellular localization assay found that TaCAMTA2-B was localized in the nucleus. In addition, self-activation experiment in the yeast indicated that TaCAMTA2-B has transcriptional activation activity. The exogenous hormone SA, ABA, JA treatments showed that TaCAMTA2-B was up-regulated when ABA treatment, and expression of TaCAMTA2-B was detected in wheat roots, stems and leaves, especially in the roots with the highest level. The above results prove that TaCAMTA2-B may be a susceptibility-related gene involved in the interaction between wheat and stripe rust. Further exploring the possible susceptibility mechanism of wheat TaCAMTAs would provide a theoretical basis for the creation of broad-spectrum and durable wheat varieties with resistance to stripe rust.
Fusarium head blight（FHB） is a devastating disease of wheat, causing yield losses and mycotoxin contamination in harvested grain. Fusarium graminearum is one of the predominant species causing FHB in wheat. Sugar transporters have not previously been identified in F. graminearum. Here, we identified two sucrose transporter genes FgSUT1 and FgSUT2 homologous to Schizosaccharomyces pombe Sut1. We obtained the Fgsut1, Fgsut2, and Fgsut1Fgsut2 double mutants. The phenotype analysis showed that the growth rate, conidiation, sexual reproduction and DON production were not affected in the Fgsut1, Fgsut2 and Fgsut1Fgsut2 double mutants. The growth rate of Fgsut1 was significantly reduced in plates with glucose as carbon source, while the Fgsut1Fgsut2 grew slower than Fgsut1. In addition, the growth rate of Fgsut1Fgsut2 also significantly reduced when fructose or maltose was used as the carbon source. These results suggested that FgSut1 and FgSut2 were functionally redundant in carbon source utilization, but they had different roles in glucose utilization. The virulence of Fgsut2 and Fgsut1Fgsut2 was decreased on wheat spikelets and coleoptiles, indicating FgSut2 may be important for virulence, and FgSut1 and FgSut2 had redundant function in fungal pathogenicity. This study established a relationship between F. graminearum carbon source utilization, toxin synthesis and pathogenicity, and enriched the research on the carbon source utilization mechanism of F. graminearum.
In order to analyze the resistant genotypes and field application of different cultivars, tomato yellow leaf curl virus（TYLCV） resistance genes Ty-1,Ty-2, Ty-3 and Ty-3a were tested from 43 tomato cultivars through molecular marker technology. At the same time, field natural identification methods were used to verify the resistance level of different genotypes. Results based on the molecular detection showed that 31 tested cultivars contained Ty genes, which accounted for 72.09% of the total varieties; During detection of the resistant genes, Ty-1 has the highest detection rate of 65.12%, which showed that Ty-1 was the most widely used gene for tomato virus disease resistance breeding. 10 resistance genotypes were detected in all tested cultivars which contain 1-3 resistance genes respectively. The resistance performance from high to low was: Ty-1/ty-1+Ty-2/ty-2+Ty-3a/ty-3、Ty-1/Ty-1+Ty-2/ty-2+Ty-3a/ty-3a、Ty-1/Ty-1、Ty-1/ty-1+Ty-3/ty-3、 Ty-1/ty-1+Ty-3a/ty-3a、Ty-1/Ty-1+Ty-3a/ty-3a、Ty-3a/ty-3a、Ty-1/ty-1+Ty-2/ty-2、Ty-1/ty-1 and Ty-2/ty-2, respectively. Resistance of the materials showed medium resistance or susceptibility when it carried one resistant gene, whereas materials carrying two or more resistant genes had more stable resistant levels. The analysis of variance showed that the mate-rials which carried 3 resistance genes had significantly higher resistance than that of Ty-1 or Ty-2 heterozygous disease resistance, but there were no significant differences between the loci with 2 resistance genes and single gene locus. The results of identification showed that there were 6 varieties with high resistance, 9 varieties showed resistance, 9 varieties showed medium resistance and 13 varieties showed susceptibility to TYLCV. The incidence of TYLCV in the identification nurseries was 58.03%, and tomato chlorosis virus（ToCV） was 4.67%. The resistance was significantly reduced after the co-infection with TYLCV and ToCV. The phenomenon implied that complex infection of TYLCV and ToCV may have potential adverse effects on the resistance genes. The above research results provide a theoretical basis for the application of disease resistance genes.
