Rhizosphere microbial communities are known to be related to plant health; using such an association for crop management requires a better understanding of this relationship. We investigated rhizosphere microbiomes associated with Verticillium wilt symptoms in two cotton cultivars. Microbial communities were profiled by amplicon sequencing, with the total bacterial and fungal DNA quantified by quantitative polymerase chain reaction based on the respective 16S and internal transcribed spacer primers. Although the level of V. dahliae inoculum was higher in the rhizosphere of diseased plants than in the healthy plants, such a difference explained only a small proportion of variation in wilt severities. Compared to healthy plants, the diseased plants had much higher total fungal/bacterial biomass ratio, as represented by quantified total fungal or bacterial DNA. The variability in the fungal/bacterial biomass ratio was much smaller than variability in either fungal or bacterial total biomass among samples within diseased or healthy plants. Diseased plants generally had lower bacterial alpha diversity in their rhizosphere, but such differences in the fungal alpha diversity depended on cultivars. There were large differences in both fungal and bacterial communities between diseased and healthy plants. Many rhizosphere microbial groups differed in their abundance between healthy and diseased plants. There was a decrease in arbuscular mycorrhizal fungi and an increase in several plant pathogen and saprophyte guilds in diseased plants. These findings suggested that V. dahliae infection of roots led to considerable changes in rhizosphere microbial communities, with large increases in saprophytic fungi and reduction in bacterial community.

Background In our previous study, a strain EBS03 with good biocontrol potential was screened out of 48 strains of cotton endophyte Bacillus subtilis by evaluating the controlling effect against cotton Verticillium wilt. However, its mechanism for controlling Verticillium wilt remains unclear. The objective of this study was to further clarify its controlling effect and mechanism against cotton Verticillium wilt. Results The results of confrontation culture test and double buckle culture test showed that the inhibitory effects of EBS03 volatile and nonvolatile metabolite on mycelium growth of Verticillium dahliae were 70.03% and 59.00%, respectively; the inhibitory effects of sporulation and microsclerotia germination were 47.16% and 70.06%, respectively. In the greenhouse test, the EBS03 fermentation broth root irrigation had the highest controlling effect at 87.11% on cotton Verticillium wilt, and significantly promoted the growth of cotton seedlings. In the field experiment, the controlling effect of EBS03 fermentation broth to cotton Verticillium wilt was 42.54% at 60 days after cotton sowing, and the boll number per plant and boll weight in EBS03 fermentation broth seed soaking, root irrigation, and spraying treatments significantly increased by 19.48% and 7.42%, 30.90% and 2.62%, 15.99% and 9.20%, respectively. Furthermore, EBS03 improved the resistance of cotton leaves against the infection of V. dahliae, and induced the outbreak of reactive oxygen species and accumulation of callose. In addition, the results of real time fluorescent quantitative polymerase chain reaction (RT-qPCR) detection showed that EBS03 significantly induced upregulation expression level of defense-related genes PAL, POD, PPO, and PR10 in cotton leaves, enhanced cotton plant resistance to V. dahliae, and inhibited colonization level of this fungal pathogen in cotton. Conclusion Bacillus subtilis EBS03 has a good biological defense capability, which can inhibit the growth and colonization level of V. dahliae, and activate the resistance of cotton to Verticillium wilt, thus increase cotton yield.

Banana (Musa acuminate L.) is an important tropical fruit in China. In October 2020, a new leaf spot disease was observed on banana plants at an orchard of Zhenkang county (23°45ʹ23.46″ N, 98°48ʹ46.52″ E), Lincang city, Yunnan province, China. The disease incidence was about 1%. The leaf spots occurred sporadically and the percentage of the leaf area covered by lesions was less than 5%. Symptoms on the leaves were initially small, irregular, reddish-brown spots that gradually expanded to fusiform-shaped lesions with a yellow halo and eventually become necrotic, dry, and cracked. To isolate the pathogen, thirty symptomatic leaves (15 mm2) from five plants were surface disinfected in 70% ethanol (10 s) and 0.8% NaClO (2 min), rinsed in sterile water three times, and transferred to potato dextrose agar (PDA) at 28°C for 5 days. Twenty-five colonies formed on the PDA plates were white with cottony aerial mycelium, round with a light orange underside. Abundant black globular acervuli semi-immersed on PDA were observed after a week. Conidia were straight or slightly curved, clavate to spindle, five cells, four septa with dimensions of 17.49 to 34.51 µm × 4.24 to 7.28 µm (avg. 23.83 × 5.62 µm; n=50). The apical and basal cells were hyaline, whereas the three median cells were dark brown. Conidia had a single basal appendage with lengths of 2.95 to 17.7 µm (avg. 7.18 µm; n=50) and two to three apical appendages with lengths of 10.7 to 53.84 µm (avg. 17.36 µm; n=50). These morphological characteristics are consistent with those of Neopestalotiopsis spp. (Maharachchikumbura et al. 2014). To confirm species, single-spore cultures of two representative isolates CATAS-102001 and CATAS-102002 were selected for further identification. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1-α) and β-tubulin (TUB2) genes of the two isolates were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728/EF2 (O’Donnell et al. 1998; Carbone and Kohn, 1999) and T1/Bt2b (Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997), respectively, and sequenced. The sequences were deposited in GenBank (ITS: OM281005 and OM281006; TEF1-α: OM328820 and OM328821; TUB2: OM328818 and OM328819). A maximum likelihood phylogenetic tree was constructed using the MEGA 7.0 (Kumar et al. 2016) based on the concatenated sequences ITS region, EF1-α and TUB2 gene, and the cluster analysis placed the representative isolates CATAS-102001 and CATAS-102002 within a clade comprising Neopestalotiopsis clavispora. The pathogenicity of two isolates was conducted on six 7-leaf-old banana seedlings. Two leaves from each potted plants were stab inoculated by puncturing into 1-mm using a sterilized needle, and stabbing three points at both sides of leaf midrib, and then placing 10 μl conidial suspension (1×106 conidia/ml) on one side of wounded points and the other side of wounded points were inoculated with sterile water as control. Inoculated plants were kept inside a plastic bag for 72 h and maintained in the greenhouse (12 h/12 h light/dark, 28°C, 90% relative humidity). The experiments were repeated twice. Irregular necrotic lesions on inoculated leaves appeared 7 days after inoculation, whereas controls were asymptomatic. The fungus was recovered from inoculated leaves, and its taxonomy was confirmed morphologically and molecularly, fulfilling Koch’s postulates. Neopestalotiopsis clavispora has been reported to cause leaf spot on Mangifera indica (Shu et al. 2020), Macadamia integrifolia (Santos et al. 2019) and Ligustrum lucidum (Chen et al. 2020). To our knowledge, this is the first report of N. clavispora on banana in China. The identification of N. clavispora as the causal agent of the observed leaf spot disease on banana is critical to the prevention and control of this disease in the future.