Acidovorax citrulli is a seed-borne pathogen causing bacterial fruit blotch of cucurbits including melon and watermelon. We investigated the roles of quorum sensing in the wild-type group II strain Aac-5 of A. citrulli by generating aacR and aacI knockout mutants and their complementation strains. We found that twitching motility and virulence were reduced, but biofilm formation and seed attachment were increased significantly in the two mutants as compared to the wild type strain. Deletion of aacR and aacI, however, had no effect on swimming motility and polar flagella formation of Aac-5. Furthermore, deletion of aacR resulted in reduced gene expression of hrpE, hrcN and pilT, while deletion of aacI affected only the expression of hrpE and pilT, not hrcN.
Root-knot nematodes especially Meloidogyne spp. are considered as most destructive obligate parasites that substantially reduce crop yield and quality. Fosthiazate is an efficient organothiophosphate chemical with nematicidal activity against Meloidogyne spp. The present study aimed to analyze the efficacy of fosthiazate against root-knot disease in Cucumis melo var. saccharinus and its potential effects on rhizosphere microbiome and metabolites. The fosthiazate (40%) was applied two times by spraying on the day of transplanting and during the pollination period (after 31 days). Samples from treatment (fosthiazate 40%: MF) and control groups (untreated plants; MCK) were analysed through metagenomic and metabolomic profiling of rhizospheres. Results revealed that root-knot index of the MF group (9.26 ± 1.28) was significantly (p < 0.05) lower than the MCK group (22.06 ± 0.71) with a control effect of 57.85% after 31 days of the first spray, whereas fosthiazate efficacy reduced to 31.87% after 38 days of second application with significantly (p < 0.05) different root-knot index values (MF: 56 ± 1.43 and; MCK: 82.26 ± 3.87). However, Cucumis melo var. saccharinus fruit yield in both groups (MCK: 21.1 ± 0.9 and MF: 21.53 ± 0.85) showed no differences (p > 0.05). Metagenomic profiling revealed Proteobacteria, Acidobacteriota, and Firmicutes as predominant phyla and Bacillus, Sphingomonas, and Acidibacter as predominant genera in rhizosphere soil samples of both MF and MCK groups. Further, a t-test revealed higher differential enrichment of Firmicutes at phylum level and Bacillus at genus level in MF than MCK. Metabolomic profiling of rhizospheric soil revealed a total of six differential metabolites (p < 0.05), four of them (Sucrose, Hexaonic acid 1, (Z)-9-Octadecenamide 1, and Hexadecanamide) were up-regulated in MF group, whereas two of them (2,3,4-Trihydroxy-3-(Hydroxymethyl) Butanol and Sulfurous acid, 2, ethylhexylundecyl ester) were down-regulated in CK group. Our study concluded that fosthiazate exhibits a better control over the rook-knot disease in the short term and resulted in trackable changes in rhizosphere microbiome and metabolome.
Colletotrichum capsici is one of the most important pathogens on chili peppers. Unreasonable application of chemical fungicides will lead to threats to human and animal health, environmental damage, and increased fungicide resistance to pathogens. As an alternative strategy, biological control has been paid more and more attention by academics. In this study, the Yb-1 strain was isolated from healthy cucumber leaves, which could significantly restrict the mycelium growth of C. capsici and Botrytis cinerea, with inhibition rates of 93.49% and 74.03%, respectively. Strain Yb-1 was identified as Bacillus velezensis by morphological features and 16S rRNA gene, gyrA gene, and gyrB gene sequence analysis. When pepper seeds were treated with different concentrations of bacterial suspension, we found that a medium-concentration treatment (104 CFU/mL and 105 CFU/mL) could promote seed germination and growth, while high-concentration treatments (108 CFU/mL) limited seed germination and growth. In addition, the root-irrigation method, acupuncture-leaf method, and leaf-cutting method were used to evaluate the safety of strain Yb-1 on peppers. The results indicated that Yb-1 did not affect the normal growth of peppers. The results of further field trials showed that the control effect of strain Yb-1 on pepper anthracnose was 59.45%. Thus, the strain B. velezensis Yb-1 has great biocontrol potential for pepper anthracnose and promotes plant growth.
Cabbage Fusarium Wilt (CFW) is a serious disease caused by Fusarium oxysporum f. sp. conglutinans in many parts of the world. The use of chemical fungicides has placed a heavy burden on the environment and is prone to drug resistance in plant pathogens. As a method with great potential, biological control has attracted the attention of many academics both at home and abroad. In this study, we have found that strains B5 and B6 had a strong inhibitory effect on various pathogens and significantly inhibited mycelium growth. They were both identified as Bacillus velezensis by morphological features, biochemical determinations, 16S rRNA gene and gyrA gene sequence analysis. When different concentrations of bacterial suspension were applied to cabbage seeds, hypocotyl and taproot length increased to varying degrees. The in vivo results showed that B5 and B6 decreased the incidence of cabbage seedling wilt disease, with B6 performing significantly better. Furthermore, B. velezensis B6 had the ability to colonize cabbage plants and rhizosphere soil. Thus, strain B6 has great potential for biocontrol development and this research could lead to the development of a promising biological agent for CFW.
Alpinia oxyphylla Miq. is mainly distributed in Hainan, Guangdong and Guangxi provinces of China. Between July and August 2021, a leaf spot disease was observed in Ledong, Hainan Province, China (18°70′20.50″ N, 109°25′25.47″E) on A.oxyphylla. The incidence of infected leaves ranged from 8% to 10%, and the incidence rate of infected plants was about 50%. Symptoms appeared as primary yellow-brown withered spots on the diseased leaves, which further developed into irregular red-brown spots. The center of the lesions was gray-black, and the tissue was irregularly necrotic, ruptured or perforated, and there were yellow chlorotic halos around the edges of the lesions (Figure 1A). Tissues 5 mm in diameter were taken from the junction of diseased and healthy tissue for pathogen isolation, Successively, a total of 8 isolates were obtained from the affected leaves. Three single spore isolates (YZ-HN-001, YZ-HN-043 and YZ-HN-051) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate YZ-HN-001 was selected for morphological and molecular identification. On Potato Dextrose Agar(PDA), the colony was gray-white at first and gradually turned dark green to dark brown with lead gray on the back, growth was slow, and mycelium was short and dense (Figure 1B and Figure 1C). Pycnidia were epiphyllous, globose, brown (about 120-140 µm in diameter), and conidia were elliptical, colorless, single celled and smooth (8-12×4-7 µm) (Figure 1D). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), 18S rRNA gene and the actin gene (ACT) by using the primers ITS1/ITS4 (White et al. 1990), EF4/Fungi5 (Khodaparase et al. 2005) and ACT-512F/ACT-783R (Carbone and Kohn. 1999). The sequences of the amplified fragments were deposited in GenBank, the ITS sequence (ON005130, 616 bp) showed 100% identity with Phyllosticta capitalensis strain CGMCC3.14345 (JN791605.1), the 18S rRNA sequence (ON005129, 541 bp) showed 99% identity with P. capitalensis isolate MUCC0029 (AB454185.1) and the ACT sequence (ON049348, 251 bp) showed 100% identity with P. capitalensis strain DZSN202005-2 (MW533248.1). A phylogenetic analysis was conducted in MEGA X using the neighbor-joining method and showed that isolate YZ-HN-001 clustered together with P. capitalensis (Figure 2). Based on the above morphological and molecular characteristics, the isolate was determined to be P. capitalensis. Pathogenicity tests were conducted in three replicates by inoculating surface-sterilized leaves of A. oxyphylla. The leaves were wounded and inoculated with colonized PDA plugs (5×5 mm) from 15-day-old cultures. Control leaves wounded in the same way and were inoculated with sterile PDA plugs (5×5 mm). Leaves were moisturized by spraying with sterile water every three days. After 20 days at room temperature (23 to 28℃), similar symptoms were observed in the inoculated leaves as in the field (Figure 1E), but no symptoms were observed on the control leaves (Figure 1F). The same P. capitalensis was reisolated in the inoculated leaves, confirming Koch’s postulates. Phyllosticta capitalensis has been reported to cause leaf spots or black spots on various host plants around the world (Wikee et al. 2013), including on oil palm (Nasehi et al. 2020), tea plant (Cheng et al. 2019 ), and castor (Tang et al. 2020). Nevertheless, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on A. oxyphylla worldwide.
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