Isolation and screening of bacterial agents with nematicidal properties
A total of 25 bacterial isolates were isolated from the rhizosphere of nematode-infested soils in North Sinai, Cairo Governorates, Egypt. Locations of soil samples, infected plants, and number of isolated bacteria are represented in Table 1. Obtained isolates were tested for their potential to inhibit the J2 of M. incognita in vitro tests. Obtained results, after 48 h of exposure, revealed that all bacterial cultures caused mortality (significant at p < 0.05) in M. incognita J2. The percentage of mortality ranged between 44 and 100%. Isolates numbered as RF5, RG9, RG10, BO16, BO17, and BB19 exhibited the highest percentage (100%) of mortality, followed by RT14 and BG8 that recorded 93 and 88.3%, respectively. The lowest were obtained from the isolates BP23, BB18, RF7, and RG11, which recorded 44, 45, 46.3, and 46.3%, respectively (Fig. 1). The bacterial culture supernatants of the 6 selected bacterial isolates were examined for their nematicidal activities in vitro tests. Obtained results after 48 h of exposure, revealed that all culture filtrates caused mortality to M. incognita J2 (Fig. 2). The supernatant of isolate RG10 exhibited the highest mortality rate (100%), followed by BB19 (98%). The lowest mortality rate (89.33%) was obtained by the RG9 culture supernatant. There are different species capable of antagonizing the plant-parasitic root-knot nematode. The term “antagonist” is used to describe a number of microorganisms including frequent enemies such as parasitoids and predators and even microorganisms that develop extracellular hydrolytic enzymes, antibiotics, or cause systemic plant resistance (Timper 2014).
Identification of the selected isolates and phylogenetic analysis
Morphological and biochemical characteristics of the selected isolates are summarized in Table 2. The isolates RF5 and RG9 were Gram positive, whereas isolates RG10, BO16, BO17, and BB19 were Gram negative. All isolates were rod-shaped and motile and showed positive reactions for catalase, gelatin liquefaction, and casein hydrolysis and a negative reaction for the urease test. The 16S rRNA nucleotide sequence revealed that the selected isolates were closely related to Paenibacillus amylolyticus, Brevibacillus agri, Gluconobacter frateurii, Beijerinckia mobilis, Achromobacter aloeverae, and Pseudomonas stutzeri with blast identity of 99, 98, 99, 97, 95, and 98%, respectively and 100% of query average. Phylogenetic tree of the 6 bacterial strains and closely correlated strains was illustrated in Fig. 3.
Characterization of nematicidal and plant growth-promoting traits of the selected isolates
The applied bacteria were tested for the production of HCN and siderophores. Pseudomonas stutzeri demonstrated a strong capability of producing HCN, while the rest did not. As for the development of siderophores, the most productive isolate was G. frateurii (64.6 μg/ml), followed by P. stutzeri and P. amylolyticus (46.4 and 41.2 μg/ml, respectively). No siderophores were detected by the 3 other bacteria as shown in Table 3. The six isolates were also checked for quantitative test of extracellular chitinase and protease in liquid medium. Data presented in Table 3 revealed that the chitinase production varied from 27 to 51.1 U/ml, the maximum production being observed for the isolate G. frateurii (51.1 U/ml) and A. aloeverae (48 U/ml), while the lowest was observed for the isolate B. mobilis (27 U/ml). All the selected isolates secreted protease enzyme at varied levels. The maximum protease activity (388.61 U/ml) was attained by G. frateurii followed by P. amylolyticus (306.91 U/ml) and P. stutzeri (297.66 U/ml), whereas the lowest enzyme activity was observed by A. aloeverae and B. mobilis with enzyme activity (160.51 and 205.74 U/ml), respectively.
All the 6 tested bacterial isolates produced IAA in culture broth; the amount, however, varied significantly among the strains and the order was G. frateurii > P. stutzeri > P. amylolyticus > B. mobilis > A. aloeverae > B. agri. Almost all isolates were able to dissolve phosphorus in the culture. The greatest solubilization was induced by P. stutzeri (25.9 μg/ml), followed by G. frateurii (22.5 μg/ml), and the least was induced by A. aloeverae (13.6 μg/ml). Brevibacillus agri had no ability to dissolve phosphorus in culture broth (Table 3). One of the most promising alternate control methods for chemical nematicides is the application of antagonistic microorganisms, particularly those that generate lytic enzymes. Batool et al. (2013) reported that P. aeruginosa caused more than 90% mortality in in vitro tests, performed with M. javanica, due to the high secretion of chitinase. Also, Cetintas et al. (2018) reported that Paenibacillus castaneae and Mycobacterium immunogenum were effective biocontrol agents for the management of the nematode M. incognita. They added that proteases and chitinases play an important role in the degradation of the nematode cuticle and serve as nematicidal factors for biocontrol of nematode populations. Production of secondary metabolites, including siderophores, protease, HCN, and chitinase by P. fluorescens and P. putida, induce mortality in wheat cyst nematode Heterodera avenae and inhibited egg hatching as reported by Ahmed (2017). According to Tran et al. (2019), the antinematode, Bacillus megaterium strain showed a good effect on promoting pepper growth against Meloidogyne sp. through its enzymatic activities, including chitinase and protease activity. Also, Soliman et al. (2019) reported that P. aeruginosa, P. polymyxa, Lysinibacillus sphaericus, B. cereus, B. subtilis, and A. xylosoxidans produced a high yield of chitinase, chitosanase, and protease exhibited in vitro antagonism against M. incognita.
In vivo evaluation of the nematicidal activity of the selected isolates against M. incognita
Data recorded under in vivo studies (Table 4) showed that the application of all bioagent treatments (P. amylolyticus, B. agri, G. frateurii, B. mobilis, A. aloeverae, P. stutzeri and their mixture) suppressed the total numbers of M. incognita on eggplant in comparison to the nematicide, Mocap 15% (Ethoprophos) and Micronema. The reduction percentages of J2s, galls, females, egg masses, eggs per egg mass, the final populations, and nematode rates of build-up that occurred with all treatments were diminished compared to control treatment. The greatest reduction percentage of J2s was obtained from P. stutzeri (78.21 %), and mixture of bacterial bioagent treatments (77.12%), while the least was in case of B. agri (55.4%) and P. amylolyticus (72.02%). Also, treatments of A. aloeverae gave the most effect in galls, females, eggs/egg mass reduction where they caused the reduction of 82.66, 80.28, and 69.20%, respectively. As well as G. frateurii recorded the most effective egg masses, final population, and rate of build-up giving 86.04, 77.75, and 1.23, respectively. The lowest females’ reduction percentages were registered by P. amylolyticus (66%), while, B. agri recorded the lowest final population reduction percentage (56.77%), followed by P. amylolyticus (71.17%). The root-knot nematodes, Meloidogyne species, are the most important group of plant-parasitic nematodes occurring worldwide (Moens and Perry 2009). They could rapidly establish feeding sites when found around plant roots, by inducing specific developmental pathways to suppress plant defense (Gheysen and Mitchum 2019). The eggs and second stage developmental stages are the most susceptible stages of plant-parasitic nematodes to be controlled through biological control. These life stages exist on soil particles outside of the plant in the surrounded water film (hygroscopic water), which enables the antagonistic micro-organisms to come into contact, infect, and parasitize the nematodes. When these two stages of the plant-parasitic nematodes are managed, the nematodes’ life cycle would be disrupted and the nematode’s population density would be decreased, resulting in an effective management (Allen 2004).
Effect of biocontrol agent on eggplant growth
The effect of biocontrol agents on the growth of eggplant infected with M. incognita was recorded in Table 5. All the treatments improved the plant growth parameters than the control. The treatment of G. frateurii gave the highest increase in shoot length (38.15%), shoot fresh weight (169.7%), and shoot dry weight (220.9%), whereas the treatment of P. amylolyticus gave the highest increase in shoot length (36.42), followed by A. aloeverae (31.72%). Pseudomonas stutzeri recorded the highest increase in shoot fresh weight (153.56%), followed by A. aloeverae (144.05%) as well as the treatment of mixture of bacteria recorded (170.9%) increase in shoot dry weight, followed by treatments of P. amylolyticus and P. stutzeri recorded (163.63%). Also, variable responses of root growth parameters were also detected. In general, all treatments of the tested agent as well as Micronema caused remarkable increase in plant growth parameters. While the application of nematicide, mocap (Ethoprophos) significantly decreased all measured plant parameters when compared to control. These results agree with that of El-Eslamboly et al. (2019). Applied bioagent could not only promote plant growth but also improve fruit quality by increasing nutrient contents such as carbohydrate, protein, and vitamin (Rashid et al. 2016).
Effect of nematode infection and application of a biocontrol agent or pathogens on total microbial count (TMC) in eggplant rhizosphere
Microbial density as affected by nematode infection and application of pathogens were shown in Fig. 4. Results indicated that TMC was higher by the application of the bacterial isolates than that obtained under the application of nematicide. The highest increment in TMC was recorded in rhizosphere of plants treated with B. mobilis (136 × 104 cfu/g dry soil), followed by A. aloeverae and P. amylolyticus (123.7 and 117.67 × 104 cfu/g dry soil), respectively. The highest population of the rhizosphere soil in the treated plots may be due to the production of different bioactive substances by the bacterial isolates which can ultimately improve plant defense response or antagonize soil nematodes (Jiang et al. 2018).