Glomus mosseae (Gerd and Trappe) and Neemex Reduce Invasion and Development of Meloidogyne incognita

Root knot nematode infestation is causing severe losses to crop production worldwide with decrease in tolerance to other abiotic factors (Oka et al., 2000). The heavy infestation of nematodes may cause the complete failure of the crop. Infected roots are unable to utilize water, minerals and fertilizers. Meloidogyne species represent the foremost nematode dilemma in developing countries. In Pakistan, root knot nematode, M. incognita, causes damage to eggplant and other vegetables (Kayani et al., 2013, 2017; Mukhtar et al., 2013a;Hussain et al., 2016) and was found economically most important (Mukhtar et al., 2013b; Tariq-Khan et al., 2016). Associations of root knot nematodes with other soil borne pathogens aggravate crop losses (Iqbal and Mukhtar, 2014; Iqbal et al., 2014; Shahbaz et al., 2015). Many chemical nematicides are being gradually prohibited for protecting vegetable crops. Hence, the improvement of other management practices and durable methods are immediately required to minimize the use of nematicides (Martin, 2003; Mukhtar et al., 2013c, 2014, 2017a, b; Hussain et al., 2014).

Biological control of nematodes using rhizosphere micro-organisms was considered in several reviews to be a potential management tactic and an effective alternative to nematicides (Kerry, 2000; Mukhtar et al., 2013d; Hussain et al., 2017a, b; Rahoo et al., 2017). Mycorrhizal fungi perform a significant role as bio-protectant against pathogens (Naher et al., 2013). Mycorrhizal fungi and root-knot nematodes share a striking feature, which is their ability to form associations with the roots of the majority of plant species, whereas other biotrophs generally show a restricted host range (Trudgill and Block, 2001). Mycorrhizal fungus and neem product have the ability to suppress root knot nematodes in different crops including vegetables (Hasan and Khan, 2004); however, no information is available on the combined application of neemex and mycorrhizal fungus for root knot nematode management and their impact on invasion and development in eggplant. Therefore, the present study was designed to exploit the potential of neemex and mycorrhizal fungus (G. mosseae) in reducing the invasion and development of M. incognita in eggplant.

Seeds of eggplant (Solanum melongena L.) were grown under greenhouse conditions (temperature ranges from 22–28°C and 80% relative humidity) and were transferred to earthen pots at 3 to 4 leaf stage. The mycorrhizal fungus, originally isolated from the field area of University of Agriculture, Faisalabad, and identified on the basis of morphological characters, was kept as a greenhouse stock culture on maize, and was applied as mycorrhizal inoculum at sowing of the test plants (Elsen et al., 2008). The inoculum consisted of rhizosphere soil from 4 months old maize pot cultures containing spores, hyphae and heavily colonized root pieces. Mycorrhizal colonization was determined by staining the roots with an ink-vinegar solution (Vierheilig et al., 1998). Each treatment was replicated ten times. M. incognita was originally isolated from susceptible tomato cultivar and afterwards maintained as a greenhouse stock culture on eggplant. When used for inoculation, egg masses were extracted from the eggplant roots and freshly hatched second-stage juveniles were collected by using modified Baermann dishes (Hooper et al., 2005).

Eggplants were planted under greenhouse. The pots contained soil, sand and manure in a 2:1:1 ratio, a substrate shown previously to be compatible with a high nematode infection potential (Vos et al., 2012). For the mycorrhizal treatment, different dosages like 100, 150 and 200 g of rhizosphere soil colonized by mycorrhizal fungus were added. Three doses of neemex, 5, 10 and 15 g were added alone and in combination. Plants from the non-mycorrhizal control treatment received 200 g of rhizosphere soil from non-colonized maize plants. After 6 weeks, eight mycorrhizal plants were uprooted to determine mycorrhizal colonization by staining the roots with ink-vinegar (Vierheilig et al., 1998). After clearing, staining and de-staining, 20 root pieces of 1 cm were mounted on permanent slides and observed with a light microscope. The mycorrhizal colonization percentage was observed according to Chaurasia and Khare (2005). After confirmation of mycorrhizal colonization, plants were inoculated with 1000 freshly hatched M. incognita second-stage juveniles. Invasion and life stages of the nematodes that invaded the control or treated roots were visualized by acid fuchsin staining of the whole root system of all the plants (Byrd et al., 1983) followed by observation with a light microscope. The number of second-stage, third-stage, fourth-stage juveniles and females were counted in acid fuchsin stained roots at 1, 2, 3, 4 and 5 weeks after inoculation.

The data were analyzed statistically by using the Fischer analysis of variance technique using MINITAB/ STAT statistical analysis software (Minitab, 2010), and treatment means were compared using least significant difference test at 5% probability level (Steel et al., 1997).

The results showed that distinct dose levels of mycorrhizal fungus and neemex differ significantly from control treatments. At the first harvest after 1 week, minimum number of J2 invaded in combined application of MF and neemex at level 3 in which 200 g soil colonized by mycorrhizal fungus was used along with 5 g neemex (Table I). Vermiform and swollen roots, developmental stages of M. incognita were recorded in all treatments but significantly lower as compared to control. When mycorrhizal fungus was applied alone with 200 g dose level, vermiform and swollen roots were recorded 27 and 14 respectively as compared to control (240 and 80, respectively). Neemex alone also showed significant difference over control as vermiform and swollen roots were 33 and 20, respectively.

Means sharing similar letters are statistically non-significant at P<0.05; MF, Mycorrhizal fungus (Glomus mosseae).

After 2 weeks, a minimum number of vermiform, swollen and sausage shaped was observed as 3, 30.4 and 41.7 units, respectively, at level 3 in which mycorrhizal fungus and neemex was applied together as compared to control (Table II). Mycorrhizal fungus and neemex alone also showed significant difference over control. Subsequent to 3 weeks; sausage shaped, immature females, adult females and egg masses were observed significantly higher in control (Table III). Combination of mycorrhizal fungus and neemex at level 3 showed minimum number of sausage (6), immature females (4.4), adult females (19.9) and egg masses (2.8) followed by level 2 and 1 (Table III). Later than 4 weeks; vermiform (168.8), swollen (114), immature females (28.9), adult females (283.3) and egg masses (158.1) were recorded significantly higher in control (Table IV) while minimum developmental stages were recorded in MF and neemex combined treatment (level 3) as vermiform stage, swollen stage, immature females, adult females and egg masses were 8.1, 8.3, 1.4, 14.7 and 13.4 units again respectively followed by level 2 and 1. Mycorrhizal fungus and neemex alone also showed significant difference as compared to control. After 5 weeks, significantly lower number of developmental stages of M. incognita were observed in combined application of MF and neemex (level 3) as vermiform (16.2), swollen (6.9), sausage (4.7), immature females (3.6), adult females (38.4) and egg masses (27.8) followed by level 2 and 1 as compared to control in which vermiform, swollen, sausage, immature females, adult females and egg masses were 219.4, 182.7, 112.6, 51.8, 347.3 and 184.3, respectively (Table V). Minimum number of developmental stages were recorded in MF (level 1, 2, 3) and neemex alone treatments (level 1, 2, 3) as compared to control.

Means sharing similar letters are statistically non-significant at P<0.05; MF, Mycorrhizal fungi (Glomus mosseae).

Means sharing similar letters are statistically non-significant at P<0.05; MF, Mycorrhizal fungus (Glomus mosseae).

Means sharing similar letters are statistically non-significant at P<0.05; MF, Mycorrhizal fungus (Glomus mosseae).

Means sharing similar letters are statistically non-significant at P<0.05; MF, Mycorrhizal fungus (Glomus mosseae).

Data of the developmental stages of M. incognita was recorded up to 5 weeks after inoculation. Depending on environmental conditions, normally M. incognita completes its life cycle in 24 to 35 days (Ploeg and Maris, 1999). As J2 penetrated the roots, developmental life stage of RKN comprised three extra moults prior to adult stage and it takes about 2 weeks, earlier than J2 moult converted into third stage, whereas fourth stage juveniles normally appear quickly and this stage prevail for almost 1 week (Moens et al., 2009). Invasion and development rate of life stages of M. incognita was decreased in the roots of eggplant treated with mycorrhizal fungus. Developmental stages of M. incognita appear progressively lower in the roots treated with mycorrhizal fungus and neemex than control. The present findings revealed that minimum J2s were penetrated in the roots, inoculated with mycorrhizal fungus and neemex alone or in combination, and in addition, development of life stages were reduced compared to the control. Reports about mycorrhizae and neemex in combined form reduced the infection and reproduction of nematodes (Khan et al., 2015; Hol and Cook, 2005; Akhtar and Siddiqui, 2008). Symbiotic association of mycorrhizal fungus effectively reduced the infection of nematodes as reported for other pathogens (Slezack et al., 2000). Current research was focused on the phases of infection that lead the invasion and development of nematodes. It showed that lesser invasion of J2 following development was partly liable for the lengthy generation time and reduced the reproduction of nematodes in plants colonized with mycorrhizal fungus. The decline in the invasion of M. incognita might be due to the allelopathic effect of mycorrhizal fungus that affects nematode motility and food finding ability (probing) in the rhizosphere. For root invasion, M. incognita directs towards appropriate host and site of infection (Curtis et al., 2009). The application of root exudates from mycorrhized tomato plants appreciably reduced the invasion of M. incognita (Vos et al., 2012).

This study demonstrated a continuously suppressing effect of MF and neemex alone or in combined form on penetration and further development of M. incognita. J2 penetration was constantly lower in the roots colonized by mycorrhizal fungus and nematode developmental stages were also reduced. Neemex and mycorrhizal fungus can be successfully used against M. incognita invasion and development in eggplant.

We are grateful to HEC Pakistan, for funding this research conducted at University of Agriculture, Faisalabad-Pakistan.

Authors have declared no conflict of interest.