Microbiome complementation from Piper colubrinum for Foot rot disease (Phytophthora capsici) management in black pepper (Piper nigrum L.)

Abstract

The study entitled “Microbiome complementation from Piper colubrinum for foot rot disease (Phytophthora capsici) management in black pepper (Piper nigrum L.)” was carried out at the Department of Microbiology, College of Agriculture, Vellayani, Thiruvananthapuram. The research aimed to isolate and characterize culturable, and analyze the unculturable microbiome of the wild pepper; Piper colubrinum, and the cultivated Piper nigrum, and evaluation of a culturable microbial consortium or synthetic microbiome with special emphasis on suppression of foot rot disease of black pepper. Characterization of the microbiome of three Piper plant types; Piper colubrinum (wild; foot rot resistant), healthy Piper nigrum plants in a Phytophthora capsici affected field, and diseased Piper nigrum affected by foot rot/quick wilt was done from rhizospheric soil and root samples collected from Ambalavayal, Wayanad, Kerala. For each plant type, three soil and three root samples were pooled into single replicate. In the 16S rDNA metagenomic analysis, operational taxonomic units (OTUs) were mainly assigned to five dominant bacterial phyla: Proteobacteria (34.74%), Firmicutes (27.41%), Acidobacteriota (9.33%), Actinobacteriota (7.58%), and Bacteroidota (6.49%), with the remainder distributed among several other phyla. Bacillus was the only genus present across all samples, a finding supported by culture dependent assays, and was most abundant in Piper colubrinum samples. Bryobacter was also widely distributed, except in Piper nigrum roots. In 18S rDNA ITS metagenomic analysis, fungal communities were dominated by Ascomycota (83.73%), followed by Basidiomycota (8.55%), Rozellomycota (3.35%), Mortierellomycota (3.31%), and Mucoromycota (1.25%). Notably, Trichoderma asperellum, which exhibited the strongest anti-oomycete activity in both in vivo and in vitro assays, was detected exclusively in P. colubrinum and absent in P. nigrum. Rarefaction curve analysis revealed greater diversity in soil samples particularly of P. nigrum than in root samples. Comparative bacterial analysis (Venn diagram) showed the highest number of unique features in PNDS (Piper nigrum diseased soil), followed by PNHS (Piper nigrum healthy soil), PNDR (Piper nigrum diseased root), PNHR (Piper nigrum healthy root), PCHS (Piper colubrinum healthy soil), and PCHR (Piper colubrinum healthy root). For fungi, the most unique features were found in PNDS, followed by PCHS, with only eight features shared among all six groups. In the present study, fifty-one bacterial isolates and eleven fungal endophytes of Piper nigrum and twenty-two bacterial isolates and three fungal endophytes of Piper colubrinum were isolated and purified. A total of seventy-three bacterial isolates and eleven fungal endophytes from Piper species were initially evaluated for their antagonistic activity through dual culture plate assay, serving as the preliminary screening step for identifying efficient culturable microbiome candidates. From this screening, twenty bacterial isolates and seven fungal endophytes were shortlisted for further assessment using dual culture plate assay, detached leaf assay, and cut stem assay. Subsequently, their plant growth-promoting traits such as ability to grow in nitrogen-free medium, phosphorus and zinc solubilization, production of phytohormones such as indole-3-acetic acid (IAA) and gibberellic acid (GA), siderophores and hydrogen cyanide (HCN) production, ACC deaminase activity, ammonia production, and synthesis of both volatile and non-volatile organic compounds were analyzed. Seven bacterial isolates (PNS14, PNS17, PNRS3, PNRO6, PCRO5, PCRS1, PCRS5) and three fungal endophytes (PCLF1, PNRF1, PCSF2) that possessed antagonistic activity together with plant growth-promoting attributes were subjected to identification through morphological, biochemical, and molecular methods. These included PNS14 (Bacillus sp.), PNS17 (B. amyloliquefaciens), PNRS3 (B. drentensis), PNRO6 (B. subtilis), PCRO5 (B. velezensis), PCRS1 (B. subtilis), and PCRS5 (Bacillus sp.), as determined by 16S rRNA sequencing. Among the fungal endophytes identified using ITS sequencing, PCLF1 (Trichoderma asperellum) alone was selected for in vivo studies, as other species PNRF1 (Rhizoctonia solani), and PCSF2 (Fusarium fujikuroi) were supposed to be plant pathogenic, and therefore excluded from further experiments. For consortium preparation, three Piper colubrinum bacterial isolates (PCRO5, PCRS1, PCRS5) were combined to form Piper colubrinum consortium PCCB, while four Piper nigrum bacterial isolates (PNS14, PNS17, PNRS3, PNRO6) were combined to form Piper nigrum consortium PNCB. All bacterial isolates (PCCB + PNCB) along with the fungal endophyte T. asperellum (PCLF1) were used as different treatments for in vivo assays. A polybag experiment was carried out with consortia of isolates from Piper species to assess their biocontrol potential and ability to promote plant growth in black pepper. Healthy two-node cuttings treated with the consortia from Piper spp. exhibited notable improvements in biometric traits, including number of leaves, number of branches, plant height, fresh weight and dry matter accumulation, when compared to untreated controls. Moreover, growth parameters were significantly higher in cuttings inoculated with the consortium than in those treated with indole-3-butyric acid (IBA) and control. A foliar infection assay was conducted to assess the disease-suppressing potential plant associated microbes of Piper spp. using two sets of treatments: set 1, seven individual bacterial isolates were applied separately as treatments, while set 2 consisted of consortial treatments (PCCB, PCCF, PNCB, and PCCB+PNCB). Lesion development was monitored from one to seven days after inoculation (DAI) with the pathogen. In Set 1, the lowest disease index was recorded in plants treated with Bacillus sp. PCRS5 (0.08), followed by Bacillus subtilis PNRO6 (0.10). In Set 2, PCCF showed the lowest disease index (0.08), achieving 73.2 % disease suppression compared to the pathogen control, followed by PCCB and PNCB (0.11 each). Furthermore, treated plants of set 1 and set 2 were evaluated for defense-related enzyme activities, including peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, superoxide dismutase (SOD), catalase, and β-1,3-glucanase. The results showed that, following P. capsici infection, these treated plants exhibited a statistically significant (p < 0.05) increase in enzyme activity compared to their levels prior to pathogen inoculation. Plants in the treatments PCCB and PCF exhibited higher survival rates (70.2 % and 73.2 %, respectively) compared to the inoculated control, when the pathogen was administered through soil. The development of a liquid formulation of the Piper spp. consortia, coupled with an analysis of bacterial and fungal population dynamics, revealed a pronounced capacity for rapid proliferation, occurring within minutes to hours. Over the course of a six-month experimental period, population stability was consistently maintained, in formulations supplemented with 2 % glycerol, 2 % PVP and 15 mM trehalose for bacteria, while fungal populations were stably maintained in talc formulation, with the colony-forming unit (cfu/mL) counts of both bacteria and fungus being recorded. The findings of this study highlight the potential of the Piper colubrinum fungal isolate Trichoderma asperellum PCLF1, along with the Piper nigrum isolates PNRO6 (Bacillus subtilis) and PCRS5 (Bacillus sp.), in promoting plant growth, providing disease protection, and enhancing defense responses in black pepper. The microbiome profiles of healthy and diseased Piper nigrum, and Piper colubrinum emphasize the suitability of these isolates for effective management of P. capsici induced foot rot disease in black pepper.