Integrated management of Meloidogyne incognita on tomato using combinations of abamectin, Purpureocillium lilacinum, rhizobacteria, and botanicals compared with nematicide

Background: Acceptable alternative eco-friendly tools in the present study were tested to control the root-knot nematode, Meloidogyne incognita, on greenhouse-cultivated vegetables. The nematicidal effect of rhizobacteria (Pseudomonas and Serratia), egg parasitic fungus (Purpureocillium lilacinum), abamectin (Streptomyces avermitilis), and 3 botanicals (colocynth, Citrullus colocynthis; moringa, Moringa oleifera; marigold, Tagetes erecta L.) singly or in combination was tested against M. incognita, in comparison with emamectin benzoate. Results: In vitro treatments revealed that egg hatching and juvenile mortality were influenced by the type of bioagents, plant species of botanicals, and exposure time. All the tested bioagents and botanicals displayed nematicidal potential via their ovicidal and larvicidal action on egg hatching and J2 mortality of M. incognita. Three and 5 days post-treatment, abamectin and emamectin benzoate were more effective than P. lilacinum, Serratia and Pseudomonas, and C. colocynthis in inhibiting egg hatching: 96.31 and 94.88%; 95.79 and 94.05%; 94.11 and 94.46%; 85.54 and 87.28%; 88.87 and 84.30%, respectively. On the other hand, after 10 days, P. lilacinum gave the highest inhibition percentage (99.00%), followed by abamectin (89.25%). However, the difference was insignificant compared with the inhibition percentage of rhizobacteria, Serratia and Pseudomonas (88.69%; p ≤ 0.05). Moreover, juvenile mortality was 100.0, 96.80, and 91.60% after 10 days of treatment, respectively. However, botanicals showed a lower effect on egg hatching and juvenile mortality. Under greenhouse conditions, potential antagonism towards M. incognita by application the mixture of biocontrol agents and botanicals was more effective in controlling M. incognita than single treatments. Conclusions: The combination of abamectin and/or emamectin benzoate with P. lilacinum and rhizobacteria was the most effective against M. incognita, followed by rhizobacteria and P. lilacinum, not only in decreasing galls and reproduction of M. incognita but also in increasing plant growth of tomato parameters than the control. The application of various bioagents including abamectin might be a potential antagonism strategy against phytonematodes in protected agricultural areas.


Background
Economic damage and yield losses in fields of vegetables are globally caused by root-knot nematodes (RKNs), Meloidogyne spp. (Jones et al. 2013). Sedentary endoparasitic nematode, Meloidogyne incognita, has a broad host range and infects plants that belong to important plant families such as tomatoes, cucumbers, eggplants, okra, and potatoes. Galling and subsequent reproduction of RKN in roots of infected plants are the primary symptoms affecting the plant's ability to uptake water and nutrients. There is an urgent need to choose an appropriate control method to secure yield and profitable production.
Generally, phytonematodes are managed by chemical nematicides or fumigants. Nowadays, alternative strategies or tools and bioagents are used to decrease populations of Meloidogyne spp., reduce pesticide usage, and avoid problems caused by chemical pesticides such as resistant cultivars, rhizobacteria, fungi, nematodes, and botanicals. Several eco-friendly methods are effective against eggs and infective juveniles of M. incognita in vitro and under greenhouse conditions (El-Ashry et al. 2020).
Rhizobacteria is one of these new strategies used in managing plant-parasitic nematodes as an alternative to chemical nematicides (Fatima et al. 2021). Moreover, fungi and botanical extracts solely or combined might be a more robust strategy to suppress nematode populations or downregulate the nematode density at-plant, keeping the nematode population below an economic threshold (Kayani et al. 2017).
To optimize the potential benefits of the various agents, researchers have recommended applying a combination of two or more beneficial microbes in biocontrol preparation for the management of plant-parasitic nematodes (Meyer and Roberts 2002). The combination of rhizospheric microorganisms with antagonistic fungi and plant growth-promoting bacteria (PGPB) could more likely have a greater variety of traits responsible for suppressing root-knot nematode over a wide range of environmental conditions. Different fungal strains isolated from nematodes have shown to produce substances that inhibit nematode egg hatching or kill nematodes (Nitao et al. 2002). Moreover, soil-inhabiting fungi, such as Paecilomyces lilacinus, Trichoderma harzianum, Verticillium chlamydosporium, Pochonia spp., Fusarium spp., and Penicillium spp., have been proved as effective tools in killing eggs, juveniles, and females of nematodes and reduced the densities of parasitic nematodes in soil (Khan et al. 2020), particularly RKNs.
Plant extracts are compounds prepared by solventbased extraction of all parts or particular parts of the plant releasing bioactive secondary metabolites and are therefore viewed as an option for controlling plant pathogens (Rodriguez et al. 2005).
Although botanicals have an environmentally and toxicologically safe, selective, and efficacious nematicidal potential (Aoudia et al. 2012), they are generally considered to be non-persistent under field conditions and readily transformed by light, oxygen, or microorganisms into less toxic products; thus, fewer residues are expected to result from the use of these natural products .
The objective of this study was to evaluate the efficacy of application of different biocontrol agents, i.e., commercial rhizobacteria, egg parasitic fungus, and botanical extracts in comparison with chemical nematicides singly or in combination as antagonistic tools used for the control of the RKN M. incognita on tomato plants.

Methods
Source and inoculum of root-knot nematode,

Meloidogyne incognita
Identified pure inoculum of M. incognita was maintained in the greenhouse on the tomato susceptible cultivar Super Strain B for use by a single egg mass to establish a nematode population and then used as a source of inoculum. The culture was maintained in the greenhouse on the tomato, Solanum lycopersicum L., susceptible cultivar Super Strain B.
Preparation of Purpureocillium lilacinum spores (inoculation of P. lilacinum) Identified culture of Purpureocillium lilacinum AUMC 10620 was obtained from the Mycological Centre (AUMC), Assiut University, Egypt. P. lilacinum was subcultured in potato dextrose agar media. The culture plates were incubated at 25°C for 7 days for full growth. The spore suspension was prepared by scrapping the mycelial mat with a sterile glass slide using sterile water. Two layers of muslin cloth were used to filter the sporal suspension, according to Yang et al. (2015).

Aqueous extract preparation
Fresh leaves of tested Citrullus colocynthis (Colocynth), Tagetes erecta L. (marigold), and Moringa oleifera (moringa) were obtained from their natural habitats at Sharkia Governorate, Egypt. The collected leaves were washed, shade dried, ground to a fine powder in a mill, kept in cool dark conditions until extraction with distilled water using a magnetic stirrer at room temperature (ca. 25°C), and then filtered via filter paper (Whatman No. 1). The extraction ratio was 1 W: 10 V (plant: solvent). The extraction period was 2 h. The filtration was stored at − 20°C for further usage. Then, each extract was arbitrarily termed as a standard solution (S).

Laboratory experiments
Effect of rhizobacteria, botanicals, commercial bionematicide, and chemical nematicide on M. incognita egg hatching Five healthy and homogenate egg masses of M. incognita were hand-picked with fine forceps from small galls and surface sterilized in 1:500 (v/v) aqueous solution of sodium hypochlorite (Clorox) for 5 min (Haseeb et al. 2005) and added to 10 ml of prepared concentrations of commercial rhizobacteria (BECTO Grow Roots® included Serratia marcescens and Pseudomonas putida/ fluorescens) and abamectin (Vertemic® 1.8% EC), fungus, three tested botanicals, and one chemical nematicide (emamectin Benzoate). Rhizobacteria, abamectin, and chemical nematicide (emamectin benzoate) were obtained from Agricultural Research Center, Giza, Egypt. Five egg masses were added to a 10-ml suspension of commercial rhizobacteria and abamectin at a concentration of 10 8 CFU/ml in Petri dishes (10 cm in diameter). The control treatment was similarly prepared in 10 ml distilled water only. Similarly, five egg masses were added to a 10-ml suspension of P. lilacinum fungus at a concentration of 10 6 CFU/ml in Petri dishes (10 cm in diameter), and the same protocol was followed with three botanicals C. colocynthis, M. oleifera, and T. erecta. Each treatment was replicated 5 times. All treatments were incubated at 22 ± 3°C. Numbers of newly hatched juveniles were counted periodically using a research microscope (× 100 magnification) after 1, 2, 3, 5, 7, and 10 days of treatment. The hatching inhibition percentage was calculated in comparison with the control treatment, according to the following equation: Effect of rhizobacteria, botanicals, commercial bionematicide, and chemical nematicide on the survival of M. incognita juveniles In vitro, to test the toxicity of rhizobacteria, fungus (P. lilacinum), botanicals (C. colocynthis, M. oleifera, and T. erecta), and chemical nematicides against M. incognita J2, 10 ml of the recommended rate of abamectin, emamectin benzoate, stock solution of tested botanicals, rhizobacteria, and abamectin at a concentration of 10 8 CFU/ml, and P. lilacinum fungus at a concentration of 10 6 CFU/ml were evaluated. Moreover, 0.1 ml containing about 100 J2 was pipetted into each Petri dish. The control treatment consisted of the 100 IJs maintained in 10 ml distilled water alone. Each treatment was replicated 5 times, and the dishes were kept at 22 ± 3°C as optimum temperature for IJ survival (Dunphy and Webster 1986). All dishes were sealed tightly with parafilm to avoid vaporization of the solution. The periodical examination was conducted by pipetting a 0.5-ml treatment solution into a Hawksley counting slide and examined with the aid of a research microscope at × 100. The nematode juveniles showing inactive straight posture or inactive (S) posture or not showing any movement after prodding were considered dead, and any other types of movement were scored as alive (Nardo and Grewal 2003). The juvenile's mortality was recorded after 1, 2, 3, and 4 days, and the mortality percentage was calculated by the following equation: Mortality % ð Þ ¼ No:of dead juveniles=total number of juveniles ð Þ Â 100:
The pots were irrigated with water as needed and kept at an air temperature of 22 ± 3°C on a glasshouse bench. Each treatment was replicated 5 times. Seventyfive days after inoculation, plants were removed carefully from pots and soaked in distilled water for 1 h. Then, the roots were stained in phloxine B and total rootknot gall index and egg masses index (RGI and EI) were evaluated (Bridge and Page 1980). Data on plant growth parameters, including fresh root and shoot weight (g), leaves fresh weight (g), stem diameter (mm), and plant height (cm), were measured. Moreover, galling and nematode development parameters, gall diameter, and the number of egg masses/plant roots; the number of IJs/100 g; and reproduction factor (RF) (final population/initial population) were assessed. The RGI was evaluated using the following scale: 0 = no galling; 1 = 1: 2 galls; 2 = 3: 10 galls; 3 = 11: 30 galls; 4 = 31: 100 galls; and 5 = more than 100 galls (Taylor and Sasser 1978). Gall diameter was also measured at its greatest diameter. Furthermore, samples of 100 g soil were processed for nematode extraction using a combination of sieving and Baermann trays technique (Hooper, 1986). The parameters changing the percentage of increase or decrease imputed to "negative or positive" control values and the current equations were used:

Statistical analysis
Statistically, the fully randomized design was implemented for laboratory experiments, while experimental units were organized in the greenhouse experiment in a randomized full block design. Data were subjected to statistical analysis using MSTAT version 4, where analysis of variance and means was compared using Duncan's multiple range test at p ≤ 0.05 probability.

Results
In vitro assay of the tested botanicals, chemical nematicide, rhizobacteria, abamectin, and Purpureocillium lilacinum on egg mass hatching The ovicidal effects of the stock solution 10% (w/v) of tested plant extracts (marigold, moringa, and colocynth) and recommended rate (RR) of chemical nematicide (emamectin benzoate), rhizobacteria (BECTO Grow Roots® included Serratia and Pseudomonas), recommend rate of abamectin (Vertemic® 1.8% EC), and P. lilacinum fungus on egg masses of M. incognita egg at different time intervals were evaluated (Table 1). Moreover, all of the tested materials were significantly (p ≤ 0.05) reduced J2 emerging from egg masses with a highly ovicidal effect compared to distilled water (DW). In vitro pathogenicity tests illustrated different levels of ovicidal effect at different times of exposure.  93.44, 95.52; 90.84, 89.36; 89.04, 88.75; 85.00, 86.32 and 84.01, 82.54 after 1 and 2 days of exposure to P. lilacinum, colocynth, rhizobacteria, marigold, and moringa. After 3 days of exposure, abamectin, chemical nematicide (emamectin benzoate), and P. lilacinum fungus were the most effective than the botanicals. The numbers of emerging J2 were 22.80, 26.00, and 36.40, respectively.
A reversible trend was observed 10 days posttreatment, and the number of emerging M. incognita J2 increased reached 48.40, 98.20, and 145.60 in RR of abamectin compared with 52.40, 22.80, and 13.60 in Petri dishes treated with P. lilacinum 5, 7, and 10 days posttreatment.
As shown in Table 2, the mortality of second-stage juveniles of M. incognita was significantly increased (p ≤ 0.05) after being treated with chemical nematicide, plant extracts, rhizobacteria (BECTO Grow Roots®), abamectin (Vertemic® 1.8% EC), and P. lilacinum than with J2 found in DW (control). However, abamectin as well as emamectin benzoate achieved the higher mortality numbers 2 days post-treatment followed by rhizobacteria colocynth and moringa. After 3 days of treatment, there was a corresponding increase in J2 mortality in a progressive manner, and both abamectin and P. lilacinum gave the best results. It was evident that P. lilacinum treatment increased M. incognita J2 mortality from day 7 until 10 with non-significant differences with abamectin, surpassing emamectin benzoate and rhizobacteria, whereas plant extracts had the least J2 mortality. The results presented in Table 2 revealed also that the highest percentage of J2 mortality was found in P. lilacinum (100%), followed by abamectin (96.80) and rhizobacteria (91.60).
In vitro nematicidal activities of such bioagents revealed that P. lilacinum, abamectin, and rhizobacteria were the most effective in killing second-stage juveniles of RKN, M. incognita. On the other hand, colocynth plant extract was more (p ≤ 0.05) effective in killing nematodes than moringa and marigold (Table 2).

Greenhouse experiments Tomato plant growth
Changes in tomato plant growth parameters (fresh root weight, shoot weight, the weight of leaves, stem diameter, and plant height) as a result of bio-agents application against M. incognita are presented (Table 3). The 3 plants extract (C. colocynthis, 15.22 g; T. erecta L., 15.18 g; M. oleifera, 14.92 g) in fresh root weight. The curative application of abamectin, emamectin benzoate, C. colocynthis, T. erecta L., M. oleifera, Serratia and Pseudomonas, and P. lilacinum significantly promoted fresh root and shoot weight than the control. Abamectin (16.68) and emamectin benzoate resulted in a significantly induced higher plant root and shoot weight than that other tomato plant treatments, and a nonsignificant difference was observed (p ≤ 0.05) between the treatment of P. lilacinum and roots of healthy plants  (Table 3). Likewise, remarkable increases in stem diameter and plant height were recorded with all tested bioagents compared with emmectin benzoate and healthy plants.
The results revealed that all treatments significantly (p ≤ 0. 05) reduced galling (as indicated by the number of galls and root gall index, RGI) and reproduction (as indicated by the number of egg masses on roots and number of juveniles in 100 g soil) than the plants inoculated with M. incognita alone. Pots treated with abamectin surpassed those treated with emmectin benzoate, botanicals, and 2 bioagents in the suppression of IJs of M. incognita/100 g soil and number of galls. On the other hand, pots treated with the fungus, P. lilacinum ranked the next, followed by emmectin benzoate in par with rhizobacteria whereas botanicals showed the lowest nematicidal effect on the number of IJs/100 g soil and root galling and moringa being the best with nonsignificant differences (P < 0.05) were indicated compared to other extracts. Similarly, a number of egg masses and galls ≥ 4 mm were significantly diminished by all treatments and abamectin P. lilacinus being the best. Among the tested materials, the 3 bio-agents gave promising results and significantly suppressed M. incognita development and reproduction, followed by moringa plant extract whereas colocynth showed the lowest nematicidal effect.

Root and soil parameters
Root parameters (number of galls, root gall index, and number egg masses) were significantly (p ≤ 0. 05) decreased in treated pots. Individual and combined inoculations of certain bio-agents, i.e., abamectin, P. lilacinum, and rhizobacteria and emmectin benzoate, significantly decreased (P ≤ 0. 05) the number of galls, root gall index, and egg mass numbers than the positive control (Tables 4 and 5) Moreover, fewer egg masses (18.20) were produced on the root systems of tomato plants grown in pots filled with soil treated with ab+Pl, followed by eb + Pl (20.40) and ab + r (23.80), compared with tomato plants grown in pots filled with untreated soil (Table 5) with non-significant differences. On the other hand, the interaction botanical extracts were less effective but significantly decreased the number of galls (38.40), egg masses (46.00), and J2 density/100 g soil than the untreated plants. Interacting P. lilacinum fungus and 3 botanical extracts gave better results in reducing the number of galls, egg masses, and J2 density in soil than did rhizobacteria with such botanicals. Plants treated with abamectin + P. lilacinum or plus  0, no galls or egg masses; 1, 1-2 galls or egg masses; 2, 3-10 galls or egg masses; 3, 11-30 galls or egg masses; 4, 31-100 galls or egg masses; and 5, more than 100 galls or egg masses, according to the scale given by Taylor and Sasser (1978) Figures in parenthesis refer to the percentage of reduction Each value is the mean of five replicates Means in each column followed by the same letter(s) are not significantly different at 5% level of probability according to Duncan's multiple range tests Reduction% ¼ Control−treatment Control Â 100

Discussion
From the obtained results, botanicals, P. lilacinum, abamectin (a natural fermentation product of Streptomyces avermitilis), and rhizobacteria affected egg hatching of M. incognita and juvenile mortality. Under greenhouse conditions, tested materials have potentially controlled RKN M. incognita. Abmectin and P. lilacinum were superior to other treatments. P. lilacinum together with abamectin gave the best results in decreasing root galls, egg masses of RKN, M. incognita, and soil nematode population as well. Moreover, P. lilacinum together with rhizobacteria was better than the mixture of botanicals gave satisfactory results in suppressing the previous criteria.
The antimicrobial activity of P. lilacinum against RKNs was less effective on juveniles than on eggs (Holland et al. 1999). Several mechanisms for the biological management of plant-parasite nematodes by P. lilacinum have been proposed. The main mechanism of action is direct infection during the egg stage. In addition, P. lilacinum was observed to produce leucine toxins, chitinases, proteases, and acetic acid to promote the infection (Khan et al. 2004).
Paecilomyces lilacinus fungus, strain 251 decreased J2 hatching in vitro and in pot experiments. P. lilacinum significantly suppressed J 2 , eggs, or egg masses of M. incognita as well as stimulated plant growth parameters. Moreover, combining P. lilacinus with other antagonistic or biocontrol agents is able to kill J2 of M. incognita and increase control effectiveness in infested fields with nematodes.
Numerous attempts have been made to use antagonistic fungi, rhizobacteria, and botanicals to control RKNs, but biocontrol agents displayed lower efficacies when used alone than chemical nematicides (Ding et al. 2020). Therefore, more than one application or repeated applications at the most suitable time are necessary to achieve long-term control of RKN and obtain effective biological control (Anastasiadisa et al. 2008).
Several studies on the combination treatments with various practices (soil solarization, chemical nematicides, botanicals, and biological agents) were conducted to effectively control RKNs (Anastasiadisa et al. 2008 and;El-Ashry et al. 2020). Based on the present study, the combination of antagonistic or biocontrol agents reduced the number of galls, egg masses, and M. incognita population as equally effective to the chemical emamectin benzoate. So far, abamectin (S. avermitilis), P. lilacinum fungus, and rhizobacteria were the most effective biocontrol agents used against phytonematodes in vitro and under greenhouse and field conditions (Khairy et al. 2021). The present findings are consistent with the results of Wen- Kun et al. (2016). They revealed that the combination of Syncephalastrum racemosum and P. lilacinus recorded the highest ovicidal activity and reduced egg hatching and mortality rate in J 2 of M. incognita than the control. Moreover, under the pot conditions, the shoot length and plant weight were significantly stimulated and galls were significantly reduced compared to those of the untreated control of cucumber plants.
Based on the findings, the bio-agent abamectin surpassed P. lilacinum in the suppression of IJs of M. incognita in soil. The combination of abamectin plus P. lilacinum, followed by P. lilacin and rhizobacteria, has potentially controlled M. incognita under greenhouse conditions, and it possibly induced the systemic resistance of plants (Sharma et al. 2020) besides their influence on nematode reproduction.
Botanical pesticides have nematicidal properties, including repellence and denaturation of proteins, depending on the type of botanical compound and pest. In plant-parasitic nematodes such as M. incognita, the mode of action includes egg hatching inhibition and suppression of the nematode population. Their mode of action includes influencing embryonic development, killing the eggs, dissolving the egg masses (Asif et al. 2013), and affecting the presence of compounds such as isothiocyanates, thiophenes, glucosides, alkaloids, phenolics, tannins, and fatty acids (Akhtar 2000). Jidere and Oluwatayo (2018) showed that the leaf extract of moringa decreased egg hatching and increased juvenile mortality of M. incognita. Marigold extract showed high hatching inhibition on treated egg masses (Guo et al. 2020). Under greenhouse conditions, moringa suppressed galling and reproduction of M. incognita and enhanced the length and fresh weight of shoots of eggplant (Khairy et al. 2021). Inhibition of egg hatching and increasing juvenile mortality of M. incognita in vitro and reduction of galling and reproduction of such nematode in vivo strongly suggest the presence of compounds that possess ovicidal and larvicidal properties.

Conclusion
Soil drenching with abamectin (S. avermitilis), P. lilacinus, and rhizobacteria provided a significant level of control of M. incognita close to that of chemical nematicide (emmitate benzoate). The use of rhizospheric microbes, such as abamectin, rhizobacteria, and fungi, is more environmentally friendly for managing phytonematode populations than chemical nematicides. The combined use of 2 different PGPB and antagonistic fungus P. lilacinum may represent a new biocontrol strategy for IPM programs to control plant-parasitic nematodes in organic agricultural systems. Moreover, this study may provide new insights into understanding the effectiveness of the mixture of bioagents (abamectin, rhizobacteria, and P. lilacinum) and botanicals against M. incognita under greenhouse conditions.