Effect of arbuscular mycorrhizal fungi, glomus mosseae and G. intraradices and antagonistic fungi pochonia chlamydosporia var. chlamydosporia and purpureocillium lilacinum on disease indices and reproduction rate of Meloidogyne javanica in three peach rootstocks under greenhouse conditions.

Introduction: Plant-parasitic nematodes cause significant yield losses in a wide range of crops (Nicol et al. 2011). Root-knot nematodes (Meloidogyne spp.) are the most important plant-parasitic nematodes, because they widely distributed all around the world and have wide host range (Chapuis-Lardy et al. 2015). Chemical nematicide is one of the primary means of control for plant-parasitic nematodes. Due to negative impact of synthetic nematicides, it will be necessary to develop other management strategies for plant-parasitic nematodes which are safe for the environment and humans (Huang et al., 2016). Latest interest in organic farming lead to substitution for conventional nematicides by low-risk compounds such as natural products derived from plants (Oka et al. 2006). Also, biological control is an interesting option to control these nematodes (Mosahaneh et al. 2020). Effective microorganisms (EM®) consist of a mixture of live cultures of microorganisms such as photosynthetic bacteria, which they are reported to reduce the incidence of pathogenic microorganisms (Condor et al., 2007). Materials and Methods: In the present study, the aerial parts of the marigold (Tagetes erecta) were collected from Shiraz, Iran. Marigold leaves dried in shade and finely grinded using an electric grinder and a stock solution (10% w/v) was prepared. Seeds of tomato (cv. Early-Urbana) were sown in plastic pots containing 1500 g of a sterilized mixture of farm soil (sandy loam soil) and cow manure. The pots were kept under greenhouse conditions with 16:8 h light to dark photoperiod and 27 ± 4 °C. Four-leaf stage seedlings were soil-drenched (50 ml per plant) with EM® or a mixture of equal amount of EM®+ marigold leaf extract at the rate of 5, 10, 15 and 20% and simultaneously inoculated with a suspension of M. javanica (6000 eggs per pot). The experiment was carried out in a completely randomized design with five replications. Sixty days after nematode inoculation, plants were harvested and the vegetative indices including shoots length, shoot fresh and dry weight and root fresh weight and the nematode population indices including the number of eggs as described by Hussey and Barker (1973), number of galls and egg masses per root system as described by Taylor and Sasser (1978) and the number of second stage juveniles (J2s) in the pot were recorded. Finally, the reproduction factor calculated as described by Sasser and Taylor (1978). Data were subjected to one-way analysis of variance (ANOVA) for plant growth parameters and two-way ANOVA for nematode population indices using SAS 9.1 program (Statistical Analysis System Institute Inc., USA). Treatment means were compared using least significance differences (LSD) at p<0.01.Results and Discussion: Results showed that soil drenching of EM® and EM®+ marigold leaf extract increased the plant growth parameters on inoculated and non-inoculated plants as compared to control. The treatment with EM®+ marigold leaf extract at the rate of 20% was the most effective treatment and increased shoot length, shoot fresh weight and shoot dry weight of non-inoculated plants by 33, 39 and 11% respectively, as compared to non-inoculted control plants. In the case of inoculated plants, shoot length, shoot fresh weight, shoot dry weight and root fresh weight of treated plats with EM®+ marigold leaf extract at the rate of 20% were 34, 31, 15 and 11% higher than inoculated control plants. The number of eggs and egg masses per root system and the reproduction factor were significantly reduced in treated plants with EM®+ marigold leaf extract at the rate of 15 and 20%. EM®+ marigold leaf extract at the rate of 20% reduced the number of eggs, galls, egg masses per root system and the reproduction factor by 28, 40, 37 and 27% respectively, as compared to control. The lowest numbers of eggmasses were observed in the root system of tomato plants treated with EM®+ marigold leaf extract at the rate of 20%. It had significant difference than other treatments, except EM® at the rate of 20%. These data for the first time in Iran, suggest that EM® might have utility in controlling root-knot nematodes. A mixture of EM® and marigold leaf extract was more effective than alone application of EM® for control of M. javanica. Findings from this study, suggest that a mixture of equal amount of EM® and marigold leaf extract at the rate of 20% reduced M. javanica reproduction rates in tomato plants grown in greenhouse.

A pot experiment was conducted to evaluate the influence of pre-inoculation of cucumber plants with each of the three arbuscular mycorrhizal (AM) fungi Glomus intraradices, Glomus mosseae, and Glomus versiforme on reproduction of the root knot nematode Meloidogyne incognita. All three AM fungi tested significantly reduced the root galling index, which is the percentage of total roots forming galls. Numbers of galls per root system were significantly reduced only in the G. intraradices + M. incognita treatment. The number of eggs per root system was significantly decreased by AM fungus inoculation, no significant difference among the three AM fungal isolates. AM inoculation substantially decreased the number of females, the number of eggs g−1 root and of the number of eggs per egg mass. The number of egg masses g−1 root was greatly reduced by inoculation with G. mosseae or G. versiforme. By considering plant growth, nutrient uptake, and the suppression of M. incognita together, G. mosseae and G. versiforme were more effective than G. intraradices.

The study aimed to evaluate the nematicidal effect of powder extracts of different coloured radish seeds against Meloidogyne incognita on tomato (Gülizar F1, susceptibile to root-knot nematode) under controlled conditions. Extractions were obtained from radish seeds of different colours (white, black, red, yellow) by using ethanol and acetone solvents. The experiment was carried out using 2, 4 and 6 g powder/plant application of the extracts in the pot. The experiment was set up in a random plot design with 5 replication for each radish seed extract and concentration. Radish seed powder was applied one week after nematode inoculation (1000 M. incognita eggs). After 50 days, the number of galls and egg masses on the roots were counted. It was determined that radish colour, extraction solvents and concentrations of extracts differed significantly for their nematicidal effects. The mean number of galls and egg masses was found to be 56 units in the negative control. Compared to the negative control, all treatments and concentrations decreased the number of galls and egg masses. The number of galls and egg masses was lower in acetone extract than in ethanole extract. The nematicidal effect was higher in yellow and red radish seeds powder application. The highest nematicidal effect was determined at 6 g powder/plant application. While the mean number of galls was 1.4 unit in the yellow seed powder application at a concentration of 6 g/plant of the extract prepared with acetone solvent, it was found to be 3.0 units in the red seed powder application at 6 g/plant of the acetone extract. The number of egg masses was 1.0 unit in the yellow seed powder application, while it was 2.8 units in the red seed powder application at 6 g/plant of the acetone extract. The acetone extract of radish seed powder can be used as an alternative to chemicals in the root-knot nematodes control.

Objective: The nematicidal effect of milk thistle leaves and seeds prepared with different solvents on Meloidogyne incognita was investigated. Materials and Methods: Acetone, ethanol and distilled water were used. The in vitro was carried out in 6 cm petri dishes. The extractions were studied with 500 and 1000 μg/ml (ppm). The in vitro and pot experiments designed random plots with 5 replications for each extraction, solvent and concentration. Four hundred second stage juvenile larvae (J2) were used as inoculum and dead individuals were counted after 48 hours. Five days after transplantation, nematode inoculation was carried out with 500 J2 per pot. After 24 hours, 30 ml of the solution was applied to the soil at 1000 ppm concentration. The experiment was terminated for 50 days. Then, gall and egg mass counts were made. Results: In vitro, the mortality rate at 1000 ppm was found to be similar in acetone (78.0%) and ethanol (80.8%) solvents in leaf extraction, while the highest was detected in ethanol (94.0%) in the seed extract. In distilled water solvent, 68.0% mortality was determined in the leaf extract and 62.2% mortality in the seed extract. There was no statistically significant difference between the leaf and seed extracts in number of galls and egg masses. No statistical difference could be determined between the solvents in the number of egg masses in seed extraction. While the number of galls in the leaf extract was found to be higher than in acetone (8.8 unit/root) and ethanol (8.0 unit/root) in distilled water (18.0 unit/root) and the difference between them was found to be significant, no statistically significant difference in the number of egg mass between the solvents. Conclusion: It was observed that all solvents of the leaf and seed extract suppressed galls and egg masses by more than 80% compared to the control.

Arbuscular mycorrhizal fungi (AMF) can increase plant tolerance and/or resistance to pests such as the root-knot nematode Meloidogyne incognita. However, the ameliorative effects may depend on AMF species. The aim of this work was therefore to evaluate whether four AMF species differentially affect plant performance in response to M. incognita infection. Tomato plants grown in greenhouse conditions were inoculated with four different AMF isolates (Claroideoglomus claroideum, Funneliformis mosseae, Gigaspora margarita, and Rhizophagus intraradices) and infected with 100 second stage juveniles of M. incognita at two different times: simultaneously or 2 weeks after the inoculation with AMF. After 60 days, the number of galls, egg masses, and reproduction factor of the nematodes were assessed along with plant biomass, phosphorus (P), and nitrogen concentrations in roots and shoots and root colonization by AMF. Only the simultaneous nematode inoculation without AMF caused a large reduction in plant shoot biomass, while all AMF species were able to ameliorate this effect and improve plant P uptake. The AMF isolates responded differently to the interaction with nematodes, either increasing the frequency of vesicles (C. claroideum) or reducing the number of arbuscules (F. mosseae and Gi. margarita). AMF inoculation did not decrease galls; however, it reduced the number of egg masses per gall in nematode simultaneous inoculation, except for C. claroideum. This work shows the importance of biotic stress alleviation associated with an improvement in P uptake and mediated by four different AMF species, irrespective of their fungal root colonization levels and specific interactions with the parasite.

An experiment was conducted to evaluate the tolerance of micropropagated and mycorrhized alpinia plants to the parasite Meloidogyne arenaria. The experimental design was completely randomized with a factorial arrangement of four inoculation treatments with arbuscular mycorrhizal fungi (AMF) (Gigaspora albida, Claroideoglomus etunicatum, Acaulospora longula, and a non-inoculated control) in the presence or absence of M. arenaria with five replicates. The following characteristics were evaluated after 270 days of mycorrhization and 170 days of M. arenaria inoculation: height, number of leaves and tillers, fresh mass of aerial and subterranean parts, dry mass of aerial parts, foliar area, nutritional content, mycorrhizal colonization, AMF sporulation, and the number of galls, egg masses, and eggs. The results indicated a significant interaction between the treatments for AMF spore density, total mycorrhizal colonization, and nutrient content (Zn, Na, and N), while the remaining parameters were influenced by either AMF or nematodes. Plants inoculated with A. longula or C. etunicatum exhibited greater growth than the control. Lower N content was observed in plants inoculated with AMF, while Zn and Na were found in larger quantities in plants inoculated with C. etunicatum. Fewer galls were observed on mycorrhized plants, and egg mass production and the number of eggs were lower in plants inoculated with G. albida. Plants inoculated with A. longula showed a higher percentage of total mycorrhizal colonization in the presence of the nematode. Therefore, the association of micropropagated alpinia plants and A. longula enhanced tolerance to parasitism by M. arenaria.

In this study, the effects of β-amino-butyric acid (BABA) on root-knot nematode(Meloidogyne javanica) infection of cucumber and accumulation of total phenolic compounds, hydrogen peroxide and activity of some enzymes related to plant defense mechanisms, i.e., guaiacol peroxidase (GPOX), polyphenol oxidase (PPO), catalase (CAT) in cucumber roots infected with nematode were investigated. Results of this study show that treating the cucumber seedlings with the above elicitor significantly reduces the nematode infection level (the nematode galls, number of egg masses per plant and number of eggs per individual egg mass) compared to control. Additionally, treatment of cucumber roots by BABA and BABA + nematode, significantly increased peroxidase, polyphenol oxidase and catalase activities in root tissues, 1 day after nematode inoculation in comparison to nematode inoculated plants as control and sterile water-treated plants. Enzyme activities reached to a maximum level at 4, 4 and 3 days after nematode inoculation, respectively. Additionally, the amount of H2O2, a product of oxidative stress, was significantly increased in the BABA and BABA + nematode treatments in comparison to control. Such increases have occurred in two phases and maximum levels of it were observed at 5 days after inoculation. Inoculation of cucumber plants by BABA also significantly increased accumulation of total phenol in comparison to control and maximum level of it was observed at 7 days after nematode inoculation. The results suggest that the inhibitory effect of BABA on the root-knot nematode (M. javanica) may be related to its ability to enhance defense responses in the cucumber roots.

The suppressive effects of six species of entomopathogenic nematodes(EPNs) against Meloidogyne incognita infected tomato were assessed in twoseparate trials under greenhouse conditions. The tested EPN species wereHeterorhabditis bacteriophora, H. megidis, H. zealandica, Steinernema feltiae, S.glaseri and S. riobravae. In the first trial, EPNs were applied as a liquid suspensionof alive or dead infective juveniles (IJs) at rate of 5000 IJs/plant in sandy and claysoils, while in the second trial, two Galleria mellonella infected cadavers of six-dayold per plant were used in sandy soil. In the two experiments, application of EPNswas accomplished simultaneously with inoculation of M. incognita. Carbofuran as astandard synthetic nematicide was used as comparison treatment at recommendedrate of 0.2 g/plant. Control treatments received only water and M. incognita at rateof1000 IJs/plant.Two months after inoculation, galling (as indicated by number of galls/plant)and reproduction (as indicated by number of egg masses /plant) as well as damage(as indicated by fresh and dry weight of areal parts) were assessed. Data showedthat, treatment of carbofuran surpassed all other treatments in minifying galling andreproduction of M. incognita in sandy and clay soils. On the other hand, curativeapplications of alive or heat-killed IJs significantly (P≤ 0.05) diminished gallformation and egg mass production in tomato roots with slightly amelioration infresh and dry weight of tomato shoot. Steinernematid species were morecomparatively effective than heterorhabditid ones. General means for number ofgalls and egg masses for steinernematid species were 52.50 and 25.33 withpercent reduction of 55.45 and 64.49 %, respectively. Whereas, the parallel valuesfor heterorhabditid species were 69.67 and 36.33 with percent reduction of 40.88and 49.07%, respectively compared to treatment of M. incognita alone. Treatmentsof alive IJs overwhelmed those of dead IJs in decreasing number of galls (withpercent reduction of 56.53 and 39.79% respectively) and egg masses (with percentreduction of 63.16 and 50.39%, successively). Moreover, utilization of two G.mellonella infected cadavers markedly lowered number of galls and egg masses and insignificantly (P≤ 0.05) improved plant growth parameters to certain extent. General means of percentage reduction in galls and egg masses were 58.46 and 54.74%, consecutively.

Fil: del Valle, Eleodoro Eduardo. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; Argentina; Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina;

Systemic and persistent effect of neem ( Azadirachta indica) formulations against root-knot nematodes, Meloidogyne javanica and their storage life

Summary The acquired resistance of plants to root-knot nematodes ( Meloidogyne spp.) can be achieved by applying chemical inducers, such as β -aminobutyric acid (BABA). Recently, the use of metal nanoparticles with multifunctional use against plant-parasitic nematodes has been presented. In this study, silver nanoparticles (AgNPs) were synthesised using the aqueous leaf extract of Malva sylvestris . The effect of different AgNPs concentrations on hatching and mortality of the second-stage juveniles (J2) of Meloidogyne javanica was investigated in vitro . The effects of AgNPs on eggplants infected with 0, 1, 2, 4 and 8 eggs (cm 3 soil) −1 were investigated under glasshouse conditions. The combined effects of AgNPs and BABA on eggplants infected with 4 and 8 eggs (cm 3 soil) −1 were investigated in another study. The results showed that increasing the concentration of AgNPs the hatching of M. javanica decreased and the mortality of J2 increased. The number of eggs, galls, egg masses and reproduction factor in plants treated with AgNPs infected with 4 eggs (cm 3 soil) −1 decreased by 43.4, 24.7, 23.5 and 43.2%, and in plants infected with 8 eggs (cm 3 soil) −1 , decreased by 64.5, 25.5, 63.1 and 64.5%, respectively, compared to control plants. The reproduction factor in infected plants with 4 and 8 eggs (cm 3 soil) −1 that were treated with BABA (1 mM) and AgNPs decreased by 51.6 and 55.9%, respectively, compared to control plants. The results of this study demonstrated the effects of BABA, AgNPs and the combination of these two substances in reducing the damage of different inoculum densities of M. javanica in eggplant.

The plant-parasitic nematode &lt;i&gt;Nacobbus aberrans&lt;/i&gt; is an endoparasite that induces gall formation in the roots and causes severe losses to diverse crops. Some populations of this nematode show preference for certain hosts, revealing the existence of "races/groups" with different behaviour and making nematode management difficult. A possible biological control alternative to reduce the damage caused by this species may be the use of arbuscular mycorrhizal fungi (AMF). In the present work, the effect of &lt;i&gt;Glomus intraradices&lt;/i&gt; on tomato plants inoculated with the nematode at transplanting and three weeks later was tested. At 60 days, the following parameters were estimated: percentage of AMF colonization, root and aerial dry weight, number of galls and egg masses, and reproduction factor (RF=final population/initial population) of &lt;i&gt;N. aberrans&lt;/i&gt;. AMF colonization was higher in the presence of the nematode. The use of AMF favoured tomato biomass and reduced the number of galls and RF on the plants inoculated with the nematode at transplanting.

Biological control is an environmentally safe alternative in the management of nematodes. The selection of new biocontrol agents has focused on the potential of Bacillus species in in vitro and in vivo studies. In this study, twenty-three local Bacillus isolates were characterised and evaluated for metabolisable carbon source, secondary metabolite production and nematicidal action. The in vitro antagonism studies were evaluated after direct confrontation between infecting juveniles of M. enterolobii and bacterial isolates. In a greenhouse, nematicidal activity was evaluated 51 days after nematode inoculation in melon plants previously inoculated with the bacteria and cultivated in pots. The numbers of egg masses, galls, J2 and eggs in the root, J2 in the soil, the reproduction factor and the biomass yield of the plants were evaluated. From the results obtained in the laboratory experiments, it was observed that five carbon sources (D-glucose, N-acetyl-glucosamine, D-maltose, malic acid and trisodium citrate) were common to all isolates, with variation in the production of secondary metabolites. The sum of nematostatic and nematicidal effects was above 90%. In the pot experiment, a significant reduction in the number of galls was observed with the treatments LCB 03, LCB 40, LCB 45, LCB 47, LCB 51, LCB 56 and LCB 5(3) and there was no increase in plant biomass. Root protection may be associated with effective colonisation through biofilm formation and the action of metabolites with nematicide and disorientation action. However, it is necessary to carry out further studies.

The effects of treating seeds of the common bean with rhizobacteria before sowing associated with different nitrogen (N) doses on the control of the nematode Meloidogyne javanica was evaluated. The assay was carried out in a greenhouse in random blocks, with a factorial arrangement of 5 &amp;times; 5 + 1: five N doses (0, 25, 50, 100, and 200 kg/ha); five isolates: Bacillus pumilus (1, 60 and 76), Bacillus subtilis.-34, Bacillus sp.-36, and one control treatment. The plants were inoculated with 3,000 nematode eggs/pot. After 60 days the assay was evaluated. In the absence of N, B. pumilus-60 promoted a reduction of more than 50% in the number of galls and egg masses compared to the control. B. pumilus (1 and 76) and Bacillus sp.-36 reduced the number of galls in combination with increased N doses. Treatment of seeds with Bacillus in combination with N fertilization contributed to a reduction in the number of galls and nematode egg masses. B. pumilus (60 and 76) reduced the number of galls and egg masses in the absence of N, indicating a nematicidal effect. The treatments had no effect on the number of second-stage juveniles, eggs, or the plant parameters.

Resistance of cotton cultivars to root-knot nematode. Meloidogyne incognita is an important pathogen of cotton in Brazil. There is limited information on resistance to Meloidogyne incognita. An experiment was carried out under greenhouse conditions to evaluate resistance of six cotton cultivars (Coodetec 401, CNPA ITA 90, Delta Opal, IAC 23, Fiber Max 986, BRS Facual) to root-knot nematodes (Meloidogyne incognita). The experimental design was randomized complete blocks with 7 treatments (cotton cultivars and susceptible tomato as control) and 20 replications. Fifty nine days after inoculation, the plants were removed carefully from pots and the number of galls, number of egg mass, number of eggs and Pf/Pi (final population/initial population) were recorded and calculated. Although none of the materials was resistant to the nematode, the cultivar IAC 23 presented lower number of galls and egg mass when compared to the other cotton cultivars (BRS Facual, Coodetec 401, Delta Opal and Fiber Max 1986).