Induced resistance by the mutualistic endophyte, Fusarium oxysporum strain 162, toward Meloidogyne incognita on tomato

The non-pathogenic endophytic fungus, Fusarium oxysporum strain 162, originally isolated from the endorhiza of tomato roots, reduces damage caused by Meloidogyne incognita, by inhibiting juvenile penetration of and development in the root. However, little is known about the mode of action of this endophyte fungus against the nematode. This study aimed at investigating how the endophyte affects nematode motility and survival and if induced resistance plays a role in the relationship. In a previous study, F. oxysporum strain 162 decreased nematode penetration of tomato up to 60%. In experiments using a split-root chamber to test for induced resistance, nematode penetration, number of galls, and number of egg masses were investigated 2 and 5 weeks after nematode inoculation. Split-root plants treated with F. oxysporum strain 162 showed 26-45% less nematode penetration, 21-36% less galls and a 22-26% reduction in the number of egg masses in the roots not directly inoculated with the fungus when compared to untreated control plants in repeated tests. In conclusion, inoculation of tomato plants with the non-pathogenic fungal endophyte F. oxysporum strain 162 resulted in a signficant reduction of nematode infection, which was in part due to induced resistance in the first 2-3 weeks after fungal inoculation.     

The Importance of the Host Plant on the Interaction Between Root-knot Nematodes Meloidogyne spp. and the Nematophagous Fungus, Verticillium chlamydosporium Goddard

The effect of the host plant on the efficacy of Verticillium chlamydosporium as a biological control agent for root-knot nematodes was investigated in four experiments. The growth of the fungus in the rhizosphere differed significantly with different plant species, the brassicas kale and cabbage supporting the most extensive colonization. The presence of nematodes in roots increased the growth of the fungus on most plants, and this effect was associated with the emergence of egg masses on the root surface; the presence of Meloidogyne incognita did not stimulate growth of the fungus in the rhizosphere until 5 weeks after the addition of infective juveniles to soil. The susceptibility of the plant host to M. incognita attack influenced the numbers of nematode eggs parasitized by the fungus. The control of the nematode was less effective on tomato roots, which produced large galls as a result of nematode infection compared with control on potato roots where galls were smaller, despite the greater abundance of the fungus in the rhizosphere of tomato plants. In large galls, a significant proportion of the egg masses remained embedded in the roots and was isolated from the fungus which was confined to the rhizosphere. Hence, the plant species has a marked effect on the efficacy of V. chlamydosporium as a biological control agent.     

Suppression of the Root-knot Nematode Meloidogyne javanica by Alginate Pellets Containing the Nematophagous Fungi Hirsutella rhossiliensis , Monacrosporium cionopagum and M. ellipsosporum

The endoparasitic fungus Hirsutella rhossiliensis and the nematode-trapping fungi Monacrosporium cionopagum and M. ellipsosporum were formulated as hyphae in alginate pellets. In a soil microcosm experiment, dried pellets of all three fungi decreased the invasion of cabbage seedlings by the root-knot nematode Meloidogyne javanica when juvenile nematodes were placed 2 cm from roots; M. cionopagum was more effective than the other two fungi, reducing nematode invasion by 40-95% with 0.24-0.94 pellets cm - 3 of soil. In a field microplot experiment, in which neither H. rhossiliensis nor M. ellipsosporum suppressed nematodes, 0.5 pellets of M. cionopagum cm - 3 of soil suppressed M. javanica invasion of tomato seedlings by 73%. In a second microplot experiment with only M. cionopagum , again at 0.5 pellets cm - 3 of soil, the fungus suppressed the invasion of tomato seedlings whether the pellets were added 0, 5 or 14 days before planting; the population density of M. cionopagum increased to nearly 3000 propagules g - 1 of soil by day 8 and then declined to less than 300 by day 22. Enchytraeid worms were observed in and around damaged and apparently destroyed pellets in both microplot experiments. Whether enchytraeids consumed the fungi or otherwise affected biological control requires additional research.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Interactions between root-knot nematode Meloidogyne javanica and Fusarium wilt disease, Fusarium oxysporum f.sp. Melonis in different varieties of melon

Fusarium oxysporum f.sp. melonis and root-knot nematode (Meloidogyne javanica) are destructive pathogens on cucurbits in Varamin area of Iran. The interaction between two pathogens was studied on local melon cultivars, Garmsar and Sooski. Inoculum of Meloidogyne javanica was prepared on susceptible cultivar, Rutgers using single egg mass method in greenhouse. Inoculum of Fusarium oxysporum f.sp. melonis (race 1) was prepared using Richard solution. A concentration of 2 x 10(5) micro conidia of fungus and 2000, 3000, 4000, 5000 eggs of nematode was used in 1 kg of autoclaved soil. Plants were inoculated with nematode at 2-3 leave stage then with fungus 2 weeks after nematode inoculation. The experiment was conducted in factoriel design based on CRD with 20 treatments, including varieties in 2 levels (Garmsar and Sooski), nematode in 5 levels (0, 2000, 3000, 4000, 5000 eggs) and fungus in 2 levels (presence and absence) and 3 replicates. The index that evaluated were growth index including fresh and dry weight of shoot and root, height, Fusarium wilt index and root gall index. Results of this experiment showed that all of treatments comparison to control were significantly different (p = 0.05) in growth index. Combination of fungus and nematode (5000 eggs) caused the most decrease in growth index on Garmsar and Sooski.     

Interaction of Vesicular-arbuscular Mycorrhizal Fungi and Phosphorus with Meloidogyne incognita on Tomato

The influence of two vesicular-arbuscular mycorrhizal fungi and phosphorus (P) nutrition on penetration, development, and reproduction by Meloidogyne incognita on Walter tomato was studied in the greenhouse. Inoculation with either Gigaspora margarita or Glomus mosseae 2 wk prior to nematode inoculation did not alter infection by M. incognita compared with nonmycorrhizal plants, regardless of soil P level (either 3 μg [low P] or 30 μg [high P] available P/g soil). At a given soil P level, nematode penetration and reproduction did not differ in mycorrhizal and nonmycorrhizal plants. However, plants grown in high P soil had greater root weights, increased nematode penetration and egg production per plant, and decreased colonization by mycorrhizal fungi, compared with plants grown in low P soil. The number of eggs per female nematode on mycorrhizal and nonmycorrhizal plants was not influenced by P treatment. Tomato plants with split root systems grown in double-compartment containers which had either low P soil in both sides or high P in one side and low P in the other, were inoculated at transplanting with G. margarita and 2 wk later one-half of the split root system of each plant was inoculated with M. incognita larvae. Although the mycoorhizal fungus increased the inorganic P content of the root to a level comparable to that in plants grown in high P soil, nematode penetration and reproduction were not altered. In a third series of experiments, the rate of nematode development was not influenced by either the presence of G. margarita or high soil P, compared with control plants grown in low P soil. These data indicate that supplemental P (30 μ/g soil) alters root-knot nematode infection of tomato more than G. mosseae and G. margarita.     

Biological Control of Meloidogyne hapla on Alfalfa and Tomato with the Fungus Meria coniospora

This study was to determine whether Arthrobotrys flagrans, A. oligospora, and Meria coniospora would control the root-knot nematode Meloidogyne hapla on alfalfa and tomato. Alfalfa seeds were coated with a fungus-rye powder in 2% cellulose and were planted in infested soil. Three-week-old seedlings from seed treated with M. coniospora had 60% and 58% fewer galls in two experiments than did seedlings from untreated seeds. Numbers of J2 in the soil were not reduced. Plant growth did not improve. When seed of tomato were coated with M. coniospora and planted in M. hapla-infested soil, roots had 34% fewer galls and 47% fewer J2 in the soil at 28 days. After 56 days there was no reduction in J2 numbers. Plant growth did not improve. When roots of tomato transplants were dusted with M. coniospora fungus-rye powder or sprayed with a spore suspension before planting in M. hapla-infested soil, 42% and 35%, respectively, fewer galls developed in 28 days on treated roots than on roots not treated with fungus. The numbers of J2 extracted from roots or recovered from soil were not reduced, however, and plant growth did not improve.     

Recovery and Longevity of Egg Masses of Meloidogyne incognita during Simulated Winter Survival

Effects of soil matrix potential on longevity of egg masses of Meloidogyne incognita were determined during simulated winter conditions. Egg masses were recovered from isolated root fragments incubated in field soil at matrix potentials of -0.1, -0.3, -1.0, and -4.0 bars throughout winter survival periods of 10 weeks for tomato roots and 12 weeks for cotton roots. Egg masses were more superficial on cotton roots than on tomato roots and were more easily dislodged from cotton roots during recovery of root fragments by elutriation. The rate of decline in numbers of eggs and J2 per egg mass was greater in wet as compared to dry soils (P = 0.01), with the relationship between numbers of eggs and J2 per egg mass and time being best described by quadratic models. Percentage hatch of recovered eggs declines linearly with time at soil matrix potentials of -0.1 and -0.3 bars, but at -1.0 and -4.0 bars the percentage hatch of recovered eggs increased before declining. Effects of soil matrix potential on numbers of eggs per egg mass and percentage hatch of recovered eggs were consistent with previous reports that low soil moisture inhibits egg hatch before affecting egg development. Estimations of egg population densities during winter survival periods will be affected by ability to recover infected root fragments from the soil without dislodging associated egg masses. There is a need for procedures for extraction of egg masses not attached to roots from the soil.     

Mode of Parasitism of Meloidogyne and Other Nemaiode Eggs by Dactylella oviparasitica

Hyphae of Dactylella oviparasitica proliferated rapidly through MeIoidogyne egg masses, and appressoria formed when they contacted eggs. The fungus probably penetrated egg shells mechanically, although chitinase production detected in culture suggested that enzymatic penetration was also possible. In soil, D. oviparasitica invaded egg masses soon after they were deposited on the root surface and eventually parasitized most of the first eggs laid. Occasionally the fungus grew into Meloidogyne females, halting egg production prematurely. The fungus parasitized eggs in the gelatinous matrix or eggs freed from the matrix and placed on agar or in soil. Specificity in nematode egg parasitism was not displayed, for D. oviparasitica parasitized eggs of four Meloidogyne spp., Acrobeloides sp., Heterodera schachtii, and Tylenchulus semipenetrans. In tests in a growth chamber, parasitism by D. oviparasitica suppressed galling on M. incognita-infected tomato plants.     

Seed treatment and soil drench with dl-β-amino butyric acid for the suppression of Meloidogyne javanica on tomato

Due to the recent environmental concerns, increasing amounts of research have been diverted to investigating natural products for the control of nematodes. dl-β-Amino butyric acid (BABA) could play a part in limiting nematode damage to plants. In this study, different concentrations of BABA were used as soil drench and seed treatment to determine if they can control Meloidogyne javanica on tomato. In an in vitro test, BABA did not impair mobility of second-stage juveniles of the nematode but 10 and 25 mg/l concentrations reduced hatch. Both of the application methods tested (drenching soil and/or pre-treating seeds with 25 mg/l of BABA) for the treatment of nematode infested tomato plants reduced the numbers of galls and egg masses by 82 %; nematode reproduction rates on these plants were reduced to one and, compared with untreated control plants, final nematode density was decreased by nearly 87 %. Increasing BABA concentrations of the treatment solutions to 200 and 500 mg/l resulted in further reduction in nematode damage and reproduction on treated plants, although the differences between the concentrations were not significant. Compared with untreated tomato, gall and egg mass production were decreased by an average of 92 %, and reproduction rates were held below one by both the 200- and 500-mg/l BABA rates. When seeds pretreated with 25 mg/l were also soil drenched with three BABA concentrations, the effects were slightly greater than when each method was used alone. Treated plants showed slight improvement in growth and weight.     

Biological Relationship of Rotyleinchulus borealis on Several Plant Cultivars

The embryogenic development of Rolylenchulus borealis, at 24-26 C, was completed on corn, in 12-15 days, and the life-cycle of the nematode from egg to egg required 35-40 days at 20-25 C. Juveniles remained in the soil as preinfective stages for 17-19 days before becoming adults. Only immature vermiform and swollen egg-laying females were found attached to corn roots. Eggs were laid in a gelatinous matrix on the root surface; the number of eggs per egg mass was 45 ± 28 on corn roots. Bean, green pea, potato, sorghum, and sweet potato were also found to be hosts of R. borealis. The nematode established a permanent feeding site on corn root in an endodermal cell that became hypertrophied. Pericyclic cells close to the feeding site showed granular cytoplasm and nuclei with hypertrophied nucleoli. A cell wall ingrowth was also noted around the area of stylet penetration into the endodermal cell.     

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato

Purpose

Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants.

Methods

Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis.

Results

Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans.

Conclusion

Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

     

Interactions of Paecilomyces lilacinus strain 251 with the mycorrhizal fungus Glomus intraradices: Implications for Meloidogyne incognita control on tomato

The interactions of Paecilomyces lilacinus strain 251 with the arbuscular mycorrhizal fungus Glomus intraradices and their significance for the control of Meloidogyne incognita on tomato were investigated in greenhouse experiments. Application of P. lilacinus had no effect on the frequency and intensity of tomato root colonization by G. intraradices. Likewise, the decline of the nematophagous fungus densities after single application in soil was not affected by the presence of the mycorrhizal fungus. Single application of P. lilacinus, as pre-planting soil treatment, resulted in significant reduction of nematode damage. In contrast, mycorrhizal inoculation did not provide sufficient biocontrol. Combined application of the two agents did not enhance root protection compared to single treatments. Double treatment of mycorrhized seedlings with P. lilacinus, as seedling drench and pre-planting soil treatment, 4 and 1 week before transplanting, respectively, resulted in the highest reduction of the nematode damage. These results indicate the potential of the commercial P. lilacinus strain 251 and mycorrhiza for integration in nematode control strategies.     

Effect of vesicular-arbuscular mycorrhiza on survival,penetration and development of root-knot nematode in tomato

Summary Effect of mycorrhizal colonisation byGlomus fasciculatum on survival, penetration and development of the root knot nematodeMeloidogyne incognita in tomato was studied. The number of giant cells formed in mycorrhizal plants was significantly low. Mycorrhizal roots did not prevent the penetration by nematode larvae. Root extract from the mycorrhizal plants brought about 50% mortality of the nematode larvae in four days time.     

Evaluation of a Native and Introduced Isolate of Pochonia chlamydosporia against Meloidogyne javanica

Growth chamber and plastic tunnel experiments were conducted to compare the ability of a native and introduced isolate of Pochonia chlamydosporia to colonize the rhizosphere of selected plant species and survive in soil. Effects of the isolates on population density of Meloidogyne javanica and yield of tomato after single or multiple fungal applications were also determined. In growth chamber experiments, both isolates showed a similar ability to colonise the rhizosphere of selected vegetables, except for the introduced isolate, which produced more colony forming units cm^-2 of root surface on tomato and cabbage than the native one. In the tunnel house, both isolates parasitized eggs of M. javanica, and the native but not the introduced isolate increased parasitism after multiple applications. The native isolate was recovered more frequently from soil, and was a better colonizer of tomato roots than the introduced one irrespective of the number of fungal applications. Multiple fungal applications of either isolate reduced the nematode gall rating, and the native isolate also reduced the final egg population in roots. Neither isolates reduced final nematode densities in soil or affected tomato yield when compared to untreated plots.     

Detection and biocontrol potential of <Emphasis Type="Italic">Verticillium leptobactrum</Emphasis> parasitizing <Emphasis Type="Italic">Meloidogyne</Emphasis> spp.

Three isolates of Verticillium leptobactrum proceeding from egg masses of root-knot nematodes (RKN) Meloidogyne spp. and soil samples collected in Tunisia were evaluated against second-stage juveniles (J2) and eggs of M. incognita, to determine the fungus biocontrol potential. In vitro tests showed that V. leptobactrum is an efficient nematode parasite. The fungus also colonized egg masses and parasitized hatching J2. In a greenhouse assay with tomato plants parasitized by M. incognita and M. javanica, V. leptobactrum was compared with isolates of Pochonia chlamydosporia and Monacrosporium sp., introducing the propagules into nematode-free or naturally infested soils. The V. leptobactrum isolates were active in RKN biocontrol, improving plants growth with a significant increase of tomato roots length, lower J2 numbers in soil or egg masses, as well as higher egg mortalities. In a second assay with M. javanica, treatments with three V. leptobactrum isolates reduced egg masses on roots as well as the density of J2 and the number of galls. To evaluate the fungus capability to colonize egg masses a nested Real-time polymerase chain reaction (PCR) assay, based on a molecular beacon probe was used to assess its presence. The probe was designed on a V. leptobactrum ITS region, previously sequenced. This method allowed detection of V. leptobactrum from egg masses, allowing quantitative DNA and fungal biomass estimations.     

Effect of organic amendment on phenolic content of soil and plant and response ofMeloidogyne javanica and its host to related compounds

Summary Amendment of soil with margosa cake or sawdust supplemented with NPK fertilizers increased its phenolic content. The concentration of total phenols was related to the amount of amendment used and varied with the length of decomposition period. Total phenols estimated in ether extract were more in margosa cake amended soil than in sawdust amended soil. Roots of tomato plants grown in amended soil showed presence of higher quantity of total phenols than those grown in non-amended soil. Exposure of females ofMeloidogyne javanica to benzoic, phenyl butyric, phenyl acetic and cinnamic acids significantly reduced their egg laying capacity. Suppression of larval motility was one of the main direct effects of these acids on the nematode. Exposure of tomato roots to different concentrations of phenyl acetic, benzoic, phenyl butyric and cinnamic acids imparted some resistance to invasion by the nematode. In such treated plants fewer larvae could penetrate the roots and develop into mature females and fewer eggs were produced. Research paper No.1455 through the Experiment Station G.B.P.U,A, & T., Pantnagar     

Influence of Calonectria crotalariae on Reproduction of Heterodera glycines on Soybean

Calonectria crotalariae enhanced root penetration of Lee 74 (susceptible) and Centennial (resistant) soybeans by juveniles of race 3 of Heterodera glycines. Numbers of cysts in and on the roots of Lee 74 increased during the first 30 days in the presence of the fungus. Percentage of root infection by the fungus increased at 40 days in Lee 74 in the presence of the nematode. Numbers of cysts in soil at 80 and 120 days after inoculation with both organisms accounted for the significantly increased nematode population levels on Lee 74. In the presence of the fungus on the resistant cultivar, significantly increased levels of cysts were recovered from soil at 120 days. Fungus infection of Centennial roots also infected with the nematode increased from 58 to 86% at 120 days. An inoculum timing study in which Lee 74 was infested with the nematode and fungus individually, sequentially, and in combination at days 0 and 35 indicated that enhanced nematode reproduction was related more to early plant-fungus than to early plant-fungus-nematode interaction(s).     

Impact of Paecilomyces lilacinus Inoculum Level and Application Time on Control of Meloidogyne incognita on Tomato

Microplot experiments were conducted to evaluate the effects of inoculum level and time of application of Paecilomyces lilacinus on the protection of tomato against MeIoidogyne incognita. The best protection against M. incognita was attained with 10 and 20 g of fungus-infested wheat kernels per microplot which resulted in a threefold and fourfold increase in tomato yield, respectively, compared with tomato plants treated with this nematode alone. Greatest protection against this pathogen was attained when P. lilacinus was delivered into soil 10 days before planting and again at planting. Yield was increased twofold compared with yield in nematode-alone plots and plots with M. incognita plus the fungus. Percentages of P. lilacinus-infected egg masses were greatest in plots treated at midseason or at midseason plus an early application, compared with plots treated with the fungus 10 days before planting and (or) at planting time.     

Effect of Meloidogyne incognita and Importance of the Inoculum on the Yield of Eggplant

The relationship between population densities of race 1 of Meloidogyne incognita and yield of eggplant was studied. Microplots were infested with finely chopped nematode-infected pepper roots to give population densities of 0, 0.062, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, and 128 eggs and juveniles/cm³ soil. Both plant growth and yield were suppressed by the nematode. A tolerance limit of 0.054 eggs and juveniles/cm³ soil and a minimum relative yield of 0.05 at four or more eggs and juveniles/cm³ soil were derived by fitting the data with the equation y = m + (1 - m)z^P⁻T. Maximum nematode reproduction rate was 12,300. Hatch of eggs from egg masses in water or from sodium hypochlorite dissolved egg masses was similar (41% and 39%), but egg viability was significantly greater from egg masses in water (58%) than from sodium hypochlorite dissolved egg masses (12%) after 4 weeks. Greater numbers of nematodes were collected from roots of tomatoes from soil infested with entire egg masses than from tomato roots from soil infested with egg masses dissolved by sodium hypochlorite.