Analysis of Bioactive Compounds Produced by Bacillus mojavensis ZA1 and Their Antagonistic Effect on Colletotrichum coccodes by GC-MS.

The plant disease Colletotrichum coccodes, which lowers potato yields, poses a severe danger to the booming potato industry. Isolated plant endophytic bacteria from highland pasture can produce a variety of metabolites that lessen the risk that the pathogen C. coccodes poses to plant growth and development. Therefore, the objective of our work was to assess substances with antipathogenic properties made by the endophytic bacteria Bacillus mojavensis ZA1. Gas chromatography-mass spectrometry (GC-MS) was used in our investigation to accomplish a thorough structural elucidation of the antipathogenic compounds produced by the endophytic bacterial strain B. mojavensis ZA1. The results showed that the metabolites extracted from ethyl acetate as an extractant were the most effective in inhibiting the pathogen C. coccodes, with 60.95% inhibition. Thirty-five distinct chemicals, including acids, esters, ketones, alcohols, amino acid ammonium salts, cyclic ethers, aromatic hydrocarbons, and heterocyclic compounds, were among the metabolites that may inhibit C. coccodes. Further analysis of the chemical groups in the compound structures revealed the potential of driving groups, such as hydroxyl, carbonyl, ester, benzene, carbon-carbon double bonds, and carbon rings, that prevent C. coccodes from performing its function. This study opens up new opportunities for plant protection programs by demonstrating that natural chemicals produced by B. mojavensis ZA1 can be used as candidates for cutting-edge plant disease management treatments.

In vitro application of proline in potato tubers under newly emerging bacteria Lelliottia amnigena infection.

Biotic stress deleteriously affects growth, development, and productivity in plants. Proline (Pro) plays a significant role in enhancing plant resistance to pathogen infection. However, its effects on reducing Lelliottia amnigena-induced oxidative stress in potato tubers remain unknown. The present study aims to evaluate the in vitro Pro treatment in potato tubers exposed to a newly emerging bacterium, L. amnigena. Sterilized healthy potato tubers were inoculated with 0.3 mL of L. amnigena suspension (3.69 × 107 CFU mL-1) 24 h before Pro (5.0 mM) application. The L. amnigena treatment significantly increased the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in the potato tubers by 80.6 and 85.6%, respectively, compared to the control. Application of proline (Pro) decreased MDA and H2O2 contents by 53.6 and 55.9%, respectively, compared to the control. Application of Pro to L. amnigena-stressed potato tubers increased the activities of NADPH oxidase (NOX), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumaryl-CoA ligase (4CL) and cinnamate-4-hydroxylase (C4H) C4H by 94.2, 96.3, 97.3, 97.1, 96.6, 79.3, 96.4, 93.6, and 96.2%, respectively, compared to the control. In comparison to the control, the genes PAL, SOD, CAT, POD, and NOX were significantly increased in the Pro-treated tubers at 5.0 mM concentration. Tubers treated with Pro + L. amnigena increased the transcript levels of PAL, SOD, CAT, POD, and NOX by 2.3, 2.2, 2.3, 2.5, and 2.8-fold respectively, compared to the control. Our findings suggested that pretreatment of tubers with Pro might reduce lipid peroxidation and oxidative stress by enhancing enzymatic antioxidant activity and gene expression.

Isolation and identification of Tussilago farfara leaf spot caused by Alternaria alternata in China.

Tussilago farfara is of vital medical value. A new leaf spot disease was observed on T. farfara leaves, in Dingxi, Gansu Province, China, in October 2019. In order to research the pathogen, the diseased samples were collected for isolation and identification. The isolate KD3 was verified by pathogenicity test, as the pathogen causing the T. farfara leaf spot disease. Its morphological characteristics were consistent with Alternaria alternata, the colony color gray-green with concentric rings, conidia fusiform and pear-shaped, brown, with 1-7 septa and 0-3 longitudinal septa, conidia size (19. 62-44.49) µm × (6.97-10.53) µm, beak length (1.35-10.03) µm × (1.01-3.63) µm, and the spore phenotype was a dwarf tree-like chain of short conidia. Multilocus sequences analysis manifested that the internal transcribed spacer (ITS), Alternaria major allergen (Alta1), and Calcium barine (CAL) sequences of strain KD3 were most closely to A. alternata (A23), with the homology of 99.47%, 99.56% and 98.28%, respectively. Based on morphological and molecular characteristics, strain KD3 was identified as A. alternata. OA was the optimal medium for its growth and PCA medium was the optimal for sporulation. This is the first report of A. alternata causing T. farfara leaf spots in China.

Potential of an endophytic bacteria Bacillus amyloliquefaciens 3-5 as biocontrol agent against potato scab.

To obtain a potential biocontrol agent for potato scab, 75 endophytic bacteria were isolated from the healthy potato tubers and strain 3-5 was selected as an optimal antagonistic bacterium against Streptomyces griseoplanus (Streptacidiphilus griseoplanus) causing potato scab. Strain 3-5 was identified as Bacillus amyloliquefaciens based on its morphological characteristics, 16S rDNA and gyrB gene sequence analysis. B. amyloliquefaciens 3-5 has biological functions of indole-3-acetic acid (IAA) production and nitrogen fixation. Polymerase chain reaction (PCR) detection revealed that B. amyloliquefaciens 3-5 had 6 diverse antibacterial substance synthesis genes, named bacD, bacAB, ituD, ituC, sfP and albF, which resulted in the production of bacilysin, iturin, surfactin and subtilosin. Field efficacy evaluation revealed that B. amyloliquefaciens 3-5 (solid fermentation) was successful in controlling potato scab with a 38.90 ± 3.2140% efficiency which is higher than other chemical bactericides except zhongshengmycin·oligosaccharins and kasugamycin·zhongshengmycin. The endophytic bacterium B. amyloliquefaciens 3-5 could be used as a biocontrol agent against potato scab due its control efficacy and environmental safety.

Identification and biological characterization of a new pathogen that causes potato scab in Gansu Province, China.

Potato scab caused by pathogenic Streptomyces is a serious soil-borne disease on potato. In this study, a new Streptomyces strain 5A-1 was isolated from potato samples in China. Based on morphological characteristics, 16S rDNA gene sequence analyses, it was identified as Streptomyces griseoplanus (Streptacidiphilus griseoplanus), pathogenicity of which was measured by the methods of small potato chips, radish slices and potato pot trial inoculation. Moreover, the pathogenic genes txtAB and tomA from the Streptomyces pathogenicity island (PAI) were detected. Determination of biological characteristics showed that the optimal temperature for the growth of S. griseoplanus strain 5A-1 was 25 °C, the optimal light condition was darkness, the optimal pH value was 8.5 and the most preferred carbon source and nitrogen source is glucose and aspartate, respectively. To our knowledge, it is the first report for S. griseoplanus, as a new pathogen, to cause potato scab.

First report of Boeremia exigua var. exigua causing branch blight on walnut in China.

Walnut (Juglans sinensis L.) is an important economic tree. Its fruit are rich in omega-3 essential fatty acids, which are valuable nutritionally (Cheon et al, 2013). In March 2019, severe branch blight of walnuts (cv. Qingxiang) were observed in two fields in Ganquan Town, Gansu Province, China (N 33°56'/E105°44'). The incidence was about 3% among 10,000 walnuts. Disease symptoms included fusiform or oval black lesions gradually expanded on the branches, blight and dieback of branches, reddish brown dead branch bark with lots of black small spots (pycnidia), and defoliation. To isolate pathogen, 30 diseased tissues collected from different trees were disinfected with 0.1% HgCl solution for 1 min, rinsed in sterilized water 3 times, and placed on potato dextrose agar (PDA) at 25℃ in the dark. About three days later, 4 fungal isolates (3-3, 3-6, 3-9 and H3) with similar morphological characteristics were obtained. Their colonies, with regular margin on OA, 6.1~6.8 cm diam. after 7 days, were loose, greenish olivaceous to olivaceous grey, velvety, floccose to woolly. The conidia (n=60) were 4.77 to 8.84 µm long (mean = 6.88 µm; SD ± 0.91 µm) × 1.71 to 3.89 µm wide (mean = 2.81 µm; SD ± 0.53 µm), cylindrical, ellipsoidal to oblong, hyaline and aseptate. Pycnidia (n=25) were 76.66 ~ 132.86µm diam. (mean = 102.93 µm; SD ± 12.15 µm), variable in shape and size, mostly globose to subglobose. These characteristics were similar to B. exigua var. exigua (Boeremia et al, 2004). Pathogenicity tests of four isolates were performed 3 times on 5 healthy 2-3 years old walnuts (cv. Qingxiang). Plants were wounded by insect needle No.6 and inoculated with 5 mm mycelium block grew on PDA medium or PDA medium as control and kept moist in climatic cabinet (> 85% RH, 25°C). After 3 days, oval black lesions were occurred on branches and gradually expanded, but control was asymptomatic. And original isolates were re-isolated from these diseased shoots. Genomic DNA of four isolates were extracted, and the internal transcribed spacer (ITS), ß-tubulin (tub2) and RNA polymerase II second largest subunit (rpb2) gene were amplified and sequenced using the primers ITS1/ITS4, Btub2Fd/Btub4Rd and RPB2-5F2/fRPB2-7CR (White et al, 1990; Woudenberg et al, 2009; Chen et al, 2015), respectively. Sequences were deposited in GenBank (accession no. ITS: MT154621, MT154622, MT154623, MT154624; tub2: MT223481, MT223482, MT223483, MT223484; rpb2: MW448152, MW448153, MW459982, MW459983), and compared with available sequences in NCBI. Results showed that ITS of four isolates have 100% sequence identity to Boeremia spp., tub2 and rpb2 have 100% sequence identity to B. exigua var. exigua (GenBank accession no. MN983734, MN983315) and B. exigua var. linicola (GenBank accession no. MN983785, MT920619). According to host specificity (Boeremia et al, 1976). A 106 conidium/mL spore suspension of four isolates or sterile water were inoculated on stem base of two-month old flax seedlings, placed in climatic cabinet (> 85% RH, 25°C) for moisturing and repeated three times. After two weeks, all inoculated plants still were asymptomatic, indicated that four isolates aren't B. exigua var. linicola. Therefore, they were identified as B. exigua var. exigua based on morphological characteristics, molecular analysis and pathogenicity tests. To our knowledge, this is the first report of B. exigua var. exigua causing walnut branch blight worldwide, which will provide further guidance for prevention and control of walnut branch blight.

First Report of Fusarium equiseti Causing Fusarium Wilt on Potato (Solanum tuberosum) in China.

Potato (Solanum tuberosum) is one of the most economically important crops in China, containing carbohydrates, protein, fiber, numerous vitamins and minerals, and is a heart healthy food (Raidl, 2020). Potato infected by Fusarium spp. exhibits quality and yield decline, and even death. In infected plants, the upper leaves exhibit chlorosis, the lower leaves wither and the vascular bundles of stems and tubers turn yellow, and then tan to brown. In August 2018, symptomatic potato stems and roots were collected from Zhangye city, Gansu province, China. Diseased stem tissues were surface sterilized with 75% alcohol for 30 s, and then rinsed in sterile water. The tissue pieces were placed on potato dextrose agar (PDA) and incubated at 25°C in darkness. Fusarium-like colonies were consistently isolated and three monoconidial isolates were obtained. Isolate 3SMJ-2 was selected as a representative for morphological characterization, molecular analysis, and pathogenicity tests. 3SMJ-2 was inoculated in PDA liquid medium, grown on a shaker for 7 days at 25℃ to obtain a mix suspension of hypha fragments and spores (107 spores/mL). Healthy potato plants, named "Xin Daping" and were planted in pots (17 cm diameter by 12 cm) filled with 2L of sterile soil per pot. After 8 weeks, the plants were inoculated with the inoculum or distilled water. Then they were incubated in growth chambers at 25°C under a 12-h/12-h day/night potato period with 90% relative humidity for 24 h. For each treatment, 3 pots were inoculated. After 50 days, 100% of the inoculated potato plants exhibited wilt symptoms similar to those in the field but the control plants were symptomless. A Fusarium identical to strain 3SMJ-2 was re-isolated from symptomatic potato plants to fulfilling Koch's postulates. Morphological characteristics of the re-isolated strain were identical to the original isolate, which confirmed pathogenicity of strain 3SMJ-2 originally isolated from the potatoes. Colonies of 3SMJ-2 were white with short conidiophores, a few microconidia and sickle-shaped macroconidia (25.2 to 42.9× 3.1 to 4.6 µm) (n = 60) with 4~7 septa, and mostly 5 septa, after cultivated on PDA in an incubator at 25℃ for 14 days. Spherical terminal or intercalary chlamydospores were observed on the mycelium. Strain 3SMJ-2 was identified preliminarily as Fusarium sp. based on morphological characteristics (Leslie et al., 2006). Genomic DNA was extracted from 3SMJ-2 using the OMEGA Fungal DNA kit according to the manufacturer's protocol. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF) and RNA polymerase II second largest subunit (RPB2) were amplified using ITS1/ITS4 (White et al., 1990), Ef728M/Tef1R (Stepien et al., 2012) and 5F2 /7cR (O'Donnell et al., 2007), respectively. After sequencing by Beijing TSINGKE Biological Technology Co., Ltd., 3 fragments of approximately 519 bp, 587 bp and 1059 bp from the strain 3SMJ-2 were deposited in GenBank as MN420681, MW561963 and MW561964. The ITS, TEF and RPB2 sequences were 100%, 100% and 99.8% identical to those of F.equiseti (KY365589, KF499577, and MH582110). Based on the pathogenicity tests, morphological characteristics and molecular analyses, we identified the strain 3SMJ-2 as F. equiseti, the pathogen causing Fusarium wilt on potato in Zhangye City. Although, F. equiseti has been reported to cause root rot of cowpea (Li et al., 2017) and sugar beet (Cao et al., 2018) in China. To our knowledge, this is the first report confirming F. equiseti causing potato wilt in China. Potato is an economically important crop in Gansu and the occurrence of the new disease caused by F. equiseti on potato needs to be properly managed to reduce yield loss.