Antagonistic bacteria against the M. javanica were isolated from liquid vermicompost, coelomic fluid of earthworm, and vermiwash, which were chosen as the rich source of bacteria. The most effective isolates belonging to the genera Bacillus, Lysinibacillus, Pseudomonas, and Sphingobacterium. The nematicidal activity and their stimulating effects on plant growth of some isolates of these genera have already been studied and shown. However, the effects of some isolates of the present study have not been previously demonstrated.
The genus Bacillus includes many species, which mostly isolated from soil and exhibit plant-promoting traits. The controlling effects of several species including B. cereus, B. subtilis, and B. megaterium on plant-parasitic nematodes have been shown in many studies (Saikia et al. 2013), and Engelbrecht et al. (2018) summarized their usefulness as natural enemies of root-knot nematodes, their nematicidal activities, and their mode of action as biocontrol agents. In the present study, the isolates B. megaterium C3 and B. safensis VW3 improved the growth parameters of the nematode infected and healthy tomato plants. Moreover, they reduced the nematode Rf by 72.8 and 62.6% in the first screening and 47.2 and 54.5% in the second selected isolates glasshouse experiments. The extent of this effect changed in the second experiment, but significantly, both Bacillus isolates reduced the nematode indices. The second experiment can confirm that by repeating the experiment, these bacteria were still effective in controlling nematode damage.
The effect of B. safensis VW3 on the plant growth parameter and the nematode indices was slightly better than B. megaterium C3. On the other hand, both species significantly inhibited the nematode egg hatching, but only B. safensis VW3 caused J2 mortality.
Strains of B. safensis had the ability to produce amylase, chitinase, keratinase, lipase, protease, and some other enzymes (Lateef et al. 2015a). B. safensis can promote plant growth (Lateef et al. 2015b), and this feature has been shown in several studies. B. safensis R173 and B. megaterium R181 along with two other strains, which isolated from the wheat rhizosphere, were the most efficient strains on corn growth in greenhouse pot test (Akinrinlola et al. 2018). In two simultaneous studies, the nematicidal activity of B. safensis has been demonstrated. The results of an in vitro screening of plant growth-promoting rhizobacteria (PGPR) indicated that three strains Bsa25, Bsa26, and Bsa27 of B. safensis and 12 other species of Bacillus caused greater than 50% H. glycines J2 mortality. In addition, the strain B. safensis Bsa27 decreased the number of H. glycines cysts at 60 days after planting in a field experiment (Xiang et al. 2017a). In another study, Xiang et al. (2017b) reported that strains of B. safensis, such as Bsa26 caused 53.7 to 100%, mortality of M. incognita J2s, and B. safensis along with other Bacillus species showed antagonistic activity against M. incognita. As shown in this study, B. safensis isolate was able to reduce the population of root-knot nematode.
B. megaterium is an aerobic and large-cell bacterium, which was found in soil and several plant tissues as an endophyte. Some of its isolates involved in chitin degradation, nitrogen fixation, or solubilization of insoluble phosphates (Logan and De Vos 2015). An endophytic isolate of B. megaterium, which was found in root nodules of Medicago polymorpha, was able to produce indole acetic acid (IAA) (Chinnaswamy et al. 2018). In addition, an isolate of B. megaterium reduced migration of M. graminicola to the root zone of rice plants and its root penetration (Padgham and Sikora 2007). Huang et al. (2010) showed that the strain B. megaterium YMF3.25 reduced egg hatching and infection of M. incognita by producing nematicidal volatiles. The results of an experiment indicated that five isolates of Bacillus out of 34 strains of endophytic bacteria, which isolated from healthy roots of black pepper, caused 100% mortality of Meloidogyne sp. J2s in vitro. Of these selective strains, B. megaterium DS9 significantly reduced the nematode populations in soil and roots of infected pepper plants by 81.86% and 73.11%, respectively, in a greenhouse (Tran et al. 2019).
The effects of B. safensis VW3 and B. megaterium C3 on egg hatching could be related to their ability to produce chitinase. The eggshell is the only structure of the nematode that contains chitin. Both species are PGPR and can improve plant growth and are nematicidal. It has been shown that there was a positive correlation between the chitinase production and nematicidal abilities of the bacterial strains and their effect on the plant growth parameters (Abdel-Salam et al. 2018).
In the present study, 2 isolates of Lysinibacillus showed nematicidal activity. L. fusiformis C1 isolated from coelomic fluid of earthworm and Lysinibacillus sp. VW6 from vermiwash. L. fusiformis C1 caused 66 and 49.2%, and Lysinibacillus sp. VW6 81.6% and 60.3% reduction the nematode Rf, respectively in the first screening and second selected isolates glasshouse experiments. Both isolates caused the greatest death rate of M. javanica J2s in vitro by 15 and 20%, respectively, but only L. fusiformis C1 significantly inhibited the nematode egg hatching by 21% and improved the infected tomato plant growth parameters.
Several species of the genus Lysinibacillus reported having nematicidal activity. L. mangiferahumi, which was separated from the soil around the roots of mango, produced nematicidal volatile compounds against M. incognita (Yang et al. 2012). It was reported that L. macroides caused 64.8% mortality of M. incognita J2s in a laboratory trial (Xiang et al. 2017b). As far as we know, the nematicidal activity of L. fusiformis has not been reported yet. Singh et al. (2013) demonstrated that the strain L. fusiformis B-CM18, which was collected from chickpea rhizosphere and has the ability to produce chitinase, possesses antifungal activity against different fungal pathogens. L. fusiformis C1, which inhibited egg hatching of M. javanica, is likely to produce chitinase.
Another bacterium with inhibitory effect on M. javanica activities in the present study was Pseudomonas resinovorans VW4, which was isolated from vermiwash. It caused the greatest reduction in galls and egg masses number and reduced nematode Rf by 73.5 and 66.0% in the first screening and second selected isolates experiments, respectively. Moreover, it inhibited 22% egg hatch of M. javanica, but did not affect J2 mortality and the growth parameters of infected tomato plants.
Many Pseudomonas species promote plant growth, induce resistance, and have the ability to protect the plant against pathogens (Preston 2004; Osman et al. 2011). P. aeruginosa is a plant growth-promoting rhizobacterium, which its nematicidal effects on root-knot nematodes were reported. Siddiqui and Ehteshamul-Haque (2001) reported that P aeruginosa IE-6S+ reduced the population of M. javanica in soil and infected tomato roots. This isolate was able to produce hydrogen cyanide (HCN). Two other isolates of this bacterium, Pa8 and Pa9, reduced egg hatching of M. incognita, increased the tomato plant growth, decreased the galls number and nematode reproduction in a glasshouse test, and produced a great amount of HCN and IAA (Singh and Siddiqi 2010). The nematicidal effects of the Antarctic strain P. putida 1A00316 on M. incognita, showed in the pot and in vitro experiments, indicated that this strain could increase the activities of phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase as defense enzymes, and induce systematic resistance in tomato plants (Tang et al. 2014). In addition, it was shown that 7 volatile compounds of strain 1A00316 had nematicidal activity against M. incognita J2s and inhibited its egg hatching when used directly or as a fumigant (Zhai et al. 2018). Although the nematicidal potential of several species of Pseudomonas has been demonstrated, there is no report recorded concerning the nematicidal activity of P. resinovorans.
In the present study, S. daejeonense LV1, which was isolated from liquid vermicompost, showed the nematicidal effect on M. javanica and caused 71.3 and 53.4% reduction in the nematode Rf respectively in the first and second glasshouse experiments. It caused M. javanica J2 mortality and improved the growth parameters of healthy and infected tomato plants but did not affect the egg hatching. Although there are no reports concerning the nematicidal activity of S. daejeonense, the effect of other species on plant-parasitic nematodes has been demonstrated. S. nematocida, the endophyte bacterium, which has been isolated from the fresh leaf of Nicotiana tabacum in China (Liu et al. 2012), caused 100% mortality of juveniles and inhibited 100% egg hatching of M. incognita (Xi et al. 2013). In addition, it was shown that Sphingobacterium sp. CIGBTb produced chitinase and had nematicidal effects on root-knot nematodes (Sánchez Ortiz et al. 2018).