Isolation and identification of endophytic bacteria associated with kiwifruit and their biocontrol potential against Meloidogyne incognita

Abstract

Background Root-knot nematodes (RKNs) cause severe losses in kiwifruit-growing regions. The endophytic bacteria could be biological agents for nematodes management. The aim of this study was the isolation and identification of endophytic bacteria from kiwifruit orchards and the evaluation of their antagonistic ability against RKN in greenhouse conditions. Results In this study, the population of nematode and the bacterial strains were isolated from kiwifruit roots and leaves in the Mazandaran and Guilan provinces of Iran. Molecular experiments were conducted to identify and confirm the bacterial isolates and RKN species. Also, the effects of bacterial isolates on nematode reproduction factors (number of galls, egg masses, and second-stage juveniles, J2) and growth parameters of kiwifruit plants were determined. The RKN was confirmed as Meloidogyne incognita by molecular identification. Also, the endophytic bacteria were identified based on supplementary experiments and molecular analyses. A total of 31 bacterial endophytes were identified to be including 12 genera of Bacillus, Pseudomonas, Staphylococcus, Exiguobacterium, Sphingomonas, Agrobacterium, Variovorax, Pantoea, Microbacterium, Streptomyces, Chryseobacterium, and Chitinophaga. Generally, Bacillus and Pseudomonas were the dominant genera that included 29.03 and 22.58% of total isolated bacteria, respectively. In vitro screening assays, P. ananatis 121.en and P. chlororaphis 54.en displayed considerable antagonistic ability on J2 mortality of M. incognita and were selected for greenhouse surveys. The isolates displayed a significant reduction in the number of galls and egg masses on roots and juvenile’s population in pot soil. Moreover, 121.en and 54.en strains significantly increased growth parameters including root fresh weight and shoot fresh weight than the control kiwifruit seedlings. Conclusions The bacterial endophytes are safe and have a low risk of managing the RKNs and can be effective microbial bio-fertilizers for improving kiwifruit plant growth under RKNs infections.

Reaction of Commercial Cultivars of Kiwifruit to Infection by Root-knot Nematode and Its Biocontrol Using Endophytic Bacteria

“…The endophytic isolates of Priestia megaterium strain 31.en (OK560186) and Agrobacterium tumefaciens strain 19.en (OK398382) had previously been isolated from seemingly healthy kiwifruit plants in orchards of Mazandaran and Guilan provinces, Iran ( Banihashemian et al, 2022 ). Isolation of these two isolates was done according to the method of Wicaksono et al (2018) .…”

“…Preparation of nematode inoculum: The Mi population, which was previously isolated from infected roots of kiwifruit plants in the Mazandaran provinces, Iran, was used in this study (Banihashemian et al, 2022). Briefly, a single egg mass, isolated from infected kiwifruit roots, was propagated and maintained on roots of susceptible tomato plants (Early Urbana variety) under greenhouse conditions (temperature of 25°C ± 2 and RH of 70%).…”

“…Plants were watered every 7 days, and 50 days after inoculation, seedlings were uprooted, and the roots were washed gently under running water to clean off soil debris. The clean roots were stained by boiling in 0.01% acid fuchsin for 15 min, then destained in acid glycerol (100 ul of HCL in 100 ml of glycerol) for three weeks with a gentle constant shaking (80 rpm) (Atighi et al, 2021). The numbers of galls and egg masses per gram of root were counted under a stereomicroscope (Nikon SMZ800).…”

“…Soil-borne pathogens cause a significant yield loss on kiwifruit annually and among them, root-knot nematodes (RKN), Meloidogyne spp., are one of the most important pathogens. Four species of RKN, including M. incognita , M. javanica , M. arenaria , and M. hapla have been reported from Iran and M. incognita ( Mi ) is known as the major species infecting kiwifruit orchards of Mazandaran Province, the biggest kiwifruit production region in Iran ( Tanha Maafi and Mahdavian, 1997 ; Banihashemian et al, 2022 ). Management of nematodes is difficult because of their wide host range and survival in diverse environmental conditions.…”

“…It has been shown that treatment of kiwifruit seedlings with Pantoea ananatis and Pseudomonas chlororaphis causes a significant reduction in the number of galls and egg masses. Moreover, they significantly increased growth parameters, including root-fresh and shoot-fresh weight compared to non-treated kiwifruit seedlings ( Banihashemian et al, 2022 ).…”