Survival of Paecilomyces lilacinus in Selected Carriers and Related Effects on Meloidogyne incognita on Tomato

Laboratory and microplot experiments were conducted to determine the influence of carrier and storage of Paecilomyces lilacinus on its survival and related protection of tomato against Meloidogyne incognita. Spores of P. lilacinus were prepared in five formulations: alginate pellets (pellets), diatomaceous earth granules (granules), wheat grain, soil, and soil plus chitin. Fungal viability was high in wheat and granules, intermediate in pellets, and low in soil and chitin-amended soil stored at 25 ± 2 C. In 1985 P. lilacinus in field microplots resulted in about a 25% increase in tomato yield and 25% gall suppression, compared with nematodes alone. Greatest suppression of egg development occurred in plots treated with P. lilacinus in pellets, wheat grain, and granules. In 1986 carryover protection of tomato against M. incognita resulted in about a threefold increase in tomato fruit yield and 25% suppression of gall development, compared with plants treated with nematodes alone. Higher numbers of fungus-infected egg masses occurred in plots treated with pellets (32%) than in those treated with chitin-amended soil (24%), wheat (16%), granules (12%), or soil (7%). Numbers of fungal colony-forming units per gram of soil in plots treated with pellets were 10-fold greater than initial levels estimated at planting time in 1986.     

Enhanced nodulation and symbiotic effectiveness of Medicago truncatula when co-inoculated with Pseudomonas fluorescens WSM3457 and Ensifer (Sinorhizobium) medicae WSM419

Aims

Low numbers of rhizobia in soil or inoculants delay nodulation and decrease symbiotic legume productivity. This study investigated the effect of co-inoculation with a helper bacterium, Pseudomonas fluorescens WSM3457 on the Medicago truncatula - Ensifer (Sinorhizobium) medicae WSM419 symbiosis challenged by a low inoculum dose.

Methods

In a glasshouse experiment the effect of co-inoculation with WSM3457 on the kinetics of nodule initiation and development was assessed 5, 7, 10, 14, 17, 21, and 42 days after inoculation of M. truncatula cv. Caliph with 10³ cells/plant of E. medicae WSM419.

Results

Co-inoculated plants had enhanced rate of nodule initiation and development, greater numbers of larger crown nodules, and by day 42 accumulated more N than plants inoculated with E. medicae WSM419 alone. Nodule development was altered by co-inoculation. Approximately 25% of nodule initials on co-inoculated plants formed in closely associated pairs, young nodules were larger with multiple meristems and developed into cluster-like multi-lobed nodules compared to those on WSM419 inoculated plants. Molecular typing showed WSM3457 occupied a significant proportion of root nodules on co-inoculated plants.

Conclusion

Co-inoculation with P. fluorescens WSM3457 enhanced symbiotic effectiveness of M. truncatula when inoculated with a low inoculum dose of E. medicae WSM419.     

Use of Brassicaceous seed meals to improve seedling emergence of tomato and pepper in Pythium ultimum infested soils

Growth room studies were conducted to determine the impact of Brassicaceae seed meals on the emergence of tomato and pepper seedlings in Pythium ultimum infested soils. Pasteurised Burch sandy loam soils were amended with intact and denatured seed meal of rape seed and mustard. Brassica juncea or Brassica napus intact seed meal increased the tomato and pepper seedling emergence. Interestingly, B. napus amended soils resulted in the same seedling emergence with B. juncea regardless of their relatively lower glucosinolate content compared to mustard-based seed meals. Seedling emergence in soils amended with intact Sinapis alba seed meal was significantly the lowest for both tomato and pepper seedlings. In contrast, seedling emergence was higher in soils amended with denatured than intact S. alba seed meals suggesting some glucosinolate-related inhibitory effect on seedling emergence of both crops. Glycine max seed meal amendment improved the seedling emergence better than the control but to a lower-level when compared to glucosinolate containing seed meals. This finding suggests that even though improvement of seedling emergence of tomato and pepper in P. ultimum infested soils can be achieved using Brassicaceae seed meals, it cannot be entirely attributed to glucosinolate-related processes. These studies demonstrate that intact B. napus and B. juncea seed meals can be used to improve tomato and pepper seedling emergence in P. ultimum infested soils.     

Mentha spicata and Salvia fruticosa composts as soil amendments in tomato cultivation

Τhe potential use of the aromatic plants Mentha spicata L. (spearmint) and Salvia fruticosa Mill. (sage) as soil amendments was evaluated. For this purpose, tomato seeds were sown in pots that had been filled with composts made from these plants and mixed with soil collected from an organically cultivated tomato field. A 2?×?2?×?4 [two types of fertilizer (synthetic and organic), two types of compost (M. spicata and S. fruticosa) and four compost rates (0%, 2%, 4% and 8%, w/w)] factorial experiment was used; the experiment was conducted twice in a growth chamber and lasted 60 days. At 0, 20, 40 and 60 days, after the establishment of the experiment, the soil bacterial and fungal abundance, the growth of nitrifying bacteria, the number of emerging weeds and the shoot length of tomato plants were measured in all treatments; at the end of the experiment, the above and belowground biomass of tomato plants was also determined. Soil microbial density increased with increasing compost rate of both species; the highest fungal and bacterial densities were recorded at 40 and 60 days, after the establishment of the experiment, respectively. Nitrifying bacteria were present in all treatments and at all sampling times. Both composts had a stimulating effect on tomato growth, which was remarkably pronounced with M. spicata. In contrast, weed emergence was reduced, but only in soils amended with M. spicata. The results suggest that M. spicata compost added at a rate of 4% to 8% is a very promising soil amendment, since it stimulates tomato growth, increases soil bacterial and fungal abundance and inhibits weed emergence. Further research is needed to elucidate the mode of action of M. spicata compost, its effect under field conditions and its possible use in mixed crop, rotational crop or cover crop systems.     

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.     

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.     

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.     

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.     

An Induced Resistance Effect of Hydroxyurea on Plants Infected by Meloidogyne javanica

Aqueous solutions of hydroxyurea (HU) in various concentrations were applied as soil drenches to Meloidogyne javanica-infected plants. At a concentration of 15 ppm, the chemical hampered giant cell formation and the number of females on the roots was 20% of that of the control but the growth of the host plants was not affected. Additional HU applications after the one at infection did not add to the inhibitory effect. HU exerted its effect on M. javanica-infected tomato in five soil types. Soil temperature of 32 C neutralized the HU-induced resistance in much the same way that high temperature breaks the natural resistance in M. incognita-resistant tomato. This study provides further evidence of the role of HU as an induced resistance agent.     

Genome sequence of Ensifer medicae strain WSM1369; an effective microsymbiont of the annual legume Medicago sphaerocarpos

Ensifer medicae WSM1369 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1369 was isolated in 1993 from a nodule recovered from the roots of Medicago sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). WSM1369 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. sphaerocarpos. Here we describe the features of E. medicae WSM1369, together with genome sequence information and its annotation. The 6,402,557 bp standard draft genome is arranged into 307 scaffolds of 307 contigs containing 6,656 protein-coding genes and 79 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.     

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.     

An In Vitro Test for Temperature Sensitivity and Resistance to Meloidogyne incognita in Tomato

An in vitro root explant tissue culture technique is described for determining susceptibility of tomato (Lycopersicon esculentum Mill.) breeding lines and cultivars to the root-knot nematode Meloidogyne incognita. Root explants were taken from 2-day-old seedlings cultured for 30 days at 28 C on Gamborg''s B-5 medium with or without nematode inoculum. The remaining portion of the root and stem from the excised root explants was transferred to soil in pots and grown to maturity in the greenhouse. In vitro root explants were evaluated for growth and occurrence of juveniles, adults, and egg masses. The regenerated plants were used to produce more seed, The proposed technique is simple, reliable, and adapted to routine screening of large numbers of F₁ and F₂ samples, and it utilizes less space than tests performed on intact plants in the greenhouse or growth chamber. Evidence is presented also on the breakdown of resistance to M. incognita under high temperature stress using this in vitro root explant technique.     

Effects of Etomopathiogenic Nematodes on Meloidogyne javanica on Tomatoes and Soybeans

Two Hawaiian isolates of Steinernema feltiae MG-14 and Heterohabditis indica MG-13, a French isolate of S. feltiae SN, and a Texan isolate of S. riobrave TX were tested for their efficacy against the root-knot nematode, Meloidogyne javanica, in the laboratory and greenhouse. Experiments were conducted to investigate the effects of treatment application time and dose on M. javanica penetration in soybean, and egg production and plant development in tomato. Two experiments conducted to assess the effects of entomopathogenic nematode application time on M. javanica penetration demonstrated that a single application of 10⁴ S. feltiae MG-14 or SN infective juveniles per 100 cm³ of sterile soil, together with 500 (MG-14) or 1,500 (SN) second-stage juveniles of M. javanica, reduced root penetration 3 days after M. javanica inoculation compared to that of a water treatment. Entomopathogenic nematode infective juveniles applied to assess the effects on M. javanica egg production did not demonstrate a significant reduction compared to that of the water control treatment. There was no dose response effect by Steinernema spp. On M. javanica root penetration or egg production. Steinernema spp. did not affect the growth or development of M. javanica-infected plants, but H. indica MG-13-treated plants had lower biomass than untreated plants infected with M. javanica. Infective juveniles of S. riobrave TX, S. feltiae SN, and MG-14 but not those of H. indica MG-13 were found inside root cortical tissues of M. javanica-infected plants. Entomopathogenic nematode antagonism to M. javanica on soybean or tomato was insufficient in the present study to provide a consistent level of nematode suppression at the concentrations of infective juveniles applied.     

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.     

Detection and Investigation of Soil Biological Activity against Meloidogyne incognita

Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm³ soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm³ soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.     

A Collagenolytic Fungus,Cunninghamella elegans,for Biological Control of Plant-parasitic Nematodes

The root-galling index of tomatoes inoculated with Meloidogyne javanica was decreased 70% when collagen was used as a soil amendment (0.1% w/w) and 90% when the amendment was supplemented with the collagenolytic fungus Cunninghamella elegans. The root-galling index was reduced 80% when the fungus was homogenized in collagen culture medium and added to soil without collagen supplement. Culture filtrates of the fungus C. elegans, grown on collagen as a single source of carbon and nitrogen, immobilized M. javanica second-stage juveniles and inhibited egg hatch. Root galling was reduced when tomato plants were inoculated with filtrate-treated juveniles. Culture filtrates reduced the motility of Rotylenchulus reniformis and Xiphinema index, but they had less effect on Anguina tritici and almost no effect on Ditylenchus dipsaci. Cunninghamella elegans had collagenolytic, elastolytic, keratinolytic, and nonspecific proteolytic activities when grown on collagen media, but only chitinolytic activity when grown on chitin media.     

Optimization of Xylanase Production by Thermomyces Lanuginosus in Tomato Seed Meal Using Response Surface Methodology

Summary A 3² central composite experimental design was performed with the aim of optimizing xylanase production by Thermomyces lanuginosus grown on corn cobs in submerged cultures. Xylanase production was first tested on different nitrogen sources (tomato skin, tomato seed meal, corn steep liquor, meat peptone, bacto-tryptone and yeast extract). Tomato seed meal was the selected substrate to test the effect of two variables on xylanase production (corn cobs and tomato seed meal concentrations). A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was corn cobs 4.6% (w/v) and tomato seed meal 2.1% (w/v). The optimum conditions found were transferred to 7-l bioreactors, where activities as high as 1630 U/ml were obtained.     

Efficacy of Organic Soil Amendments for Management of Heterodera glycines in Greenhouse Experiments

In a repeated greenhouse experiment, organic soil amendments were screened for effects on population density of soybean cyst nematode (SCN), Heterodera glycines, and soybean growth. Ten amendments at various rates were tested: fresh plant material of field pennycress, marigold, spring camelina, and Cuphea; condensed distiller’s solubles (CDS), ash of combusted CDS, ash of combusted turkey manure (TMA), marigold powder, canola meal, and pennycress seed powder. Soybeans were grown for 70 d in field soil with amendments and SCN eggs incorporated at planting. At 40 d after planting (DAP), many amendments reduced SCN egg population density, but some also reduced plant height. Cuphea plant at application rate of 2.9% (amendment:soil, w:w, same below), marigold plant at 2.9%, pennycress seed powder at 0.5%, canola meal at 1%, and CDS at 4.3% were effective against SCN with population reductions of 35.2%, 46.6%, 46.7%, 73.2%, and 73.3% compared with control, respectively. For Experiment 1 at 70 DAP, canola meal at 1% and pennycress seed powder at 0.5% reduced SCN population density 70% and 54%, respectively. CDS at 4.3%, ash of CDS at 0.2%, and TMA at 1% increased dry plant mass whereas CDS at 4.3% and pennycress seed powder at 0.1% reduced plant height. For Experiment 2 at 70 DAP, amendments did not affect SCN population nor plant growth. In summary, some amendments were effective for SCN management, but phytoxicity was a concern.     

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.