Pseudomonas Species Isolated Via High-Throughput Screening

Tao et al. AMB Expr (2020) 10:193 https://doi.org/10.1186/s13568-020-01132-1 ORIGINAL ARTICLE Open Access Pseudomonas species isolated via high-throughput screening signifcantly protect cotton plants against verticillium wilt Xiaoyuan Tao1, Hailin Zhang1, Mengtao Gao2, Menglin Li2, Ting Zhao1 and Xueying Guan1* Abstract Verticillium wilt (VW) caused by Verticillium dahliae is a devastating soil-borne disease that causes severe yield losses in cotton and other major crops worldwide. Here we conducted a high-throughput screening of isolates recovered from 886 plant rhizosphere samples taken from the three main cotton-producing areas of China. Fifteen isolates dis- tributed in diferent genera of bacteria that showed inhibitory activity against V. dahliae were screened out. Of these, two Pseudomonas strains, P. protegens XY2F4 and P. donghuensis 22G5, showed signifcant inhibitory action against V. dahliae. Additional comparative genomic analyses and phenotypical assays confrmed that P. protegens XY2F4 and P. donghuensis 22G5 were the strains most efcient at protecting cotton plants against VW due to specifc biological control products they produced. Importantly, we identifed a signifcant efcacy of the natural tropolone compound 7-hydroxytropolone (7-HT) against VW. By phenotypical assay using the wild-type 22G5 and its mutant strain in 7-HT production, we revealed that the 7-HT produced by P. donghuensis is the major substance protecting cotton against VW. This study reveals that Pseudomonas specifcally has gene clusters that allow the production of efective antipath- ogenic metabolites that can now be used as new agents in the biocontrol of VW. Keywords: Rhizosphere, Pseudomonas, Verticillium wilt, Biocontrol, 7-hydroxytropolone Key points Introduction Cotton verticillium wilt (VW) is a singularly destructive • Pseudomonas spp. isolated from high-throughput fungal disease caused by Verticillium dahliae, which is screening showed the most infuential activities to regarded as “the cancer of cotton”. Te V. dahliae fungus multiple strains of V. dahlia. invades the vascular system through the roots and soon • P. protegens XY2F4 and P. donghuensis 22G5 showed causes systemic infection, leading to a series of symp- that Pseudomonas spp. have developed specifc toms including leaf chlorosis, necrosis or wilting, leaf or mechanisms against V. dahlia. boll abscission, and even plant death. VW-related dam- • 7-hydroxytropolone produced by P. donghuensis is age results in reduced cotton yield and lower fber quality the major ingredient to protect cotton against verti- in agricultural production (Wang et al. 2016). Currently, cillium wilt. around 50% of the cotton planting area in China (2.5 million hectares) is VW-infected, leading to direct economic losses of about 250–310 million USD annually (Wang et al. 2016). Disease management mainly includes crop rotation to non-host plants, fungicide fumigation and *Correspondence: [email protected] breeding of resistant cultivars (Klosterman et al. 2009). 1 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China Crop rotation is a preventative, but not curative disease Full list of author information is available at the end of the article management strategy since V. dahliae can survive for © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Tao et al. AMB Expr (2020) 10:193 Page 2 of 12 extremely long periods of time in the soil as microsclero- were cultured on Czapek agar plates at 28 °C for 4–5 days tia even in the absence of a suitable host (Termorshuizen after which 5 mL liquid Czapek was dispensed into 1995). Disease control of VW using fungicide fumigation Petri plates to collect the conidia. Te conidia suspen- is efective, but expensive and environmentally unfriendly sion was then transferred to 100 mL liquid Czapek and (Jordan 1972). Breeding of cultivars with broad-spectrum cultured for 7 days until cell density reached OD600 2, 6 = resistance is considered to be one of the most practica- contains ~ 3 × 10 conidia/mL. Finally, the conidia were ble and efective approaches. However, it is difcult to fltered through a 500-micron sieve for use in inoculation apply biotechnology to breed VW-resistant cotton due to assays. Te V. dahliae strain stock was composed of the the lack of resistance markers in cotton germplasm, only conidia suspensions with 20% glycerol. a few commercial upland cotton cultivars have developed with moderate levels of VW resistance (Zhang et al. Bacteria isolation and culture 2012a). Bacterial isolates were recovered from 886 plant rhizos- Rhizobacteria have great potential to improve sus- phere samples taken from the three main cotton-produc- tainable agricultural practices due to their infuence on ing areas of China (the Yangtze river basin, the Yellow growth, yield, nutrient uptake, and biotic/abiotic toler- River basin and Xinjiang) (Additional fle 1: Table S1). ance of crops. Benefcial rhizobacteria are able to colo- Te samples were placed in separate, labeled 50 mL tubes nize the rhizosphere (the root surface or intercellular flled with enough ddH 2O to ensure that they were com- spaces of plants), which impacts the plant by deliver- pletely submerged, and then tubes were shaken approxi- ing biocontrol and other benefcial factors (Lugtenberg mately 4–5 times to mix. 800 µL of the mixture was et al. 2001). To date, multiple isolates from genera of aliquoted for gradient dilution (10 −1, 10−2, 10−3, 10−4 and Enterobacter (Li et al. 2012a), Bacillus (Li et al. 2012b; 10−5). 1:103 or 1:104 was considered a suitable dilution Zhang et al. 2018), Serratia plymuthica (Vleesschauwer ratio and 100 µL solution was plated on LB media and 2007), Streptomyces (Xue et al. 2013) and Pseudomonas inoculated overnight at 30 °C. Plates were stored at 4 °C (Erdogan and Benlioglu 2010) have documented biocon- for 3 days in order to enhance the formation of fuores- trol activities against V. dahliae in in planta assays. Tus, cent pigments in bacterial colonies. benefcial rhizobacteria with inhibitory action against V. dahliae are promising biocontrol agents for the manage- High‑throughput screening for bacterial isolates ment of VW in cultivated cotton (Tjamos et al. 2000). with inhibitory action against V. dahliae However, the specifc mechanisms underlying the bio- A plate assay was performed to screen isolates for inhibi- control of VW have yet to be determined. In this research tory action against V. dahliae. In a 10-mL tube, 6 mL we employed a high-throughput screening for inhibitory top agar (0.8% w/v) was cooled to less than 50 °C and isolates and comparative genomic analysis to uncover the gently mixed with 60 µL V. dahliae V991 culture stock mode of action of two new Pseudomonas strains with sig- (OD600 = 2), resulting in an initial density of 0.02/mL nifcant V. dahliae inhibitory capacity. Tis study charts at OD600 in the top agar. Tis solution was then quickly a path toward the development of probiotics and active poured on top of a Czapek agar plate, gently shaken by ingredients for biocontrol agents (BCAs) to ameliorate hand in a radial/rocking manner, and allowed to solid- cotton VW disease. ify. Afterwards, 5 µL overnight culture of candidate bacterial isolates was inoculated on top of the agar plate. Materials and methods 16 candidate isolates were able to be tested per plate via Plant culture high-throughput screening. Plates were sealed with para- Upland cotton (Gossypium hirsutum) cultivars Texas flm and cultured at 28 °C. Any inhibitory action by the Marker-1 (TM-1) and Junmian1 were grown in soil con- bacterial isolates against V. dahliae was revealed by the sisting of 25% vermiculite and 75% artifcial soil at 28 °C appearance of a zone of inhibition on the agar plate. Te with a 16 h/8 h light/dark cycle in growth chambers. size of the zone of inhibition was recorded at 72 h post One-week old seedlings were used in the planta assays. inoculation and inhibitory action was qualitatively determined. For those isolates that produced a visible zone V. dahliae culture of inhibition, additional confrmational assays were per- Highly virulent strains of V. dahliae, including V07DF2, formed using other highly virulent strains of V. dahliae, V08DF2, V15QY1, and V991 were gifts from Institute of including V07DF2, V08DF2 and V15QY1. Plant Protection, Jiangsu Academy of Agricultural Sci- ences. Te highly toxic and defoliant wild type patho- 16S rRNA identifcation and species designation genic V. dahliae strain V991 was used in planta assay 16S rDNA was amplifed using the primer pair 27F and (Sun et al. 2013; Zhang et al. 2012b). V. dahlia strains 1492R (Additional fle 1: Table S2). Sequencing results Tao et al. AMB Expr (2020) 10:193 Page 3 of 12 were identifed by using BLAST to search the NCBI 16S Gene mutation rRNA database. Species was designated based on the best Construction of an in-frame deletion mutant of orf12 hit for each species in BLAST and confrmed by genome- () from the 7-HT gene cluster in Pseudomonas dongh- based taxonomy by Type Strain Genome Server (https :// uensis 22G5 was performed as has been described pre- tygs.dsmz.de) (Meierkolthof and Goker 2019).

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