Indigenous Populations of a Biological Control Agent in Agricultural Field Soils Predicted Suppression of a Plant Pathogen.

The nematophagous fungus Hyalorbilia oviparasitica and relatives (Hyalorbilia spp.) are known to parasitize several endoparasitic nematodes. In this project, we hypothesized that indigenous populations of this fungus could be used to predict nematode suppression in agricultural field soils. We quantified Hyalorbilia spp. in soil samples from 44 different sugar beet fields in the Imperial Valley of California. Seven soils harboring Hyalorbilia spp. and two that tested negative for the fungi were examined for nematode suppressive activity. Untreated and autoclaved portions of each soil were planted with cabbage and infested with sugar beet cyst nematode (Heterodera schachtii) juveniles. Females and cysts of H. schachtii were enumerated after 12 weeks. In the seven soils harboring Hyalorbilia spp., females and cysts in the untreated soils were reduced by 61 to 82% compared with the autoclaved controls. Soils with no detectable Hyalorbilia spp. exhibited no nematode suppression. Two novel Hyalorbilia strains, HsImV25 and HsImV27, were isolated from H. schachtii females reared in field soil using an enrichment and double-baiting cultivation technique. Both strains suppressed H. schachtii populations by more than 80% in soil-based assays, confirming that Hyalorbilia spp. are the likely causal agents of the nematode suppression in these soils. This study demonstrated that indigenous populations of a hyperparasite (Hyalorbilia spp.) in agricultural field soils predicted suppressive activity against a soilborne plant pathogen (H. schachtii). To our knowledge, this is the first report to demonstrate this capability. We anticipate that this research will provide a blueprint for other similar studies, thereby advancing the field of soilborne biological control.

Real-Time PCR to Phenotype Resistance to the Citrus Nematode Tylenchulus semipenetrans Cobb.

As pest management options, such as nematicides, become more restrictive, developing rootstocks resistant to the citrus nematode is fundamental for citrus production. This study provides an updated methodology to screen for citrus nematode resistance in rootstock-breeding programs. We developed a novel method to extract female citrus nematodes from roots that is suitable for molecular work and a real-time-PCR-based nematode quantification method for Tylenchulus semipenetrans. These procedures allow scaling up screening to high-throughput workflows, increasing the chances of finding rootstock candidates that combine all the desired traits. Our results contribute to the growing literature supporting quantification of nematodes with molecular methods.

The Evaluation of Egg-Parasitic Fungi Paraboeremia taiwanensis and Samsoniella sp. for the Biological Control of Meloidogyne enterolobii on Chinese Cabbage.

Meloidogyne enterolobii, an aggressive plant-parasitic nematode, has been causing great yield loss worldwide in recent years. With no resistant Chinese cabbage cultivar available currently, a biological control strategy is needed to offer an eco-friendly option for sustainable farming. In this study, the nematode suppression efficacy of two newly isolated fungi, Paraboeremia taiwanensis and Samsoniella sp., were evaluated against M. enterolobii and compared to the known biological control agents Hyalorbilia oviparasitica strain DoUCR50 and Purpureocillium lilacinum strain 251 (PL251). Both P. taiwanensis and Samsoniella sp. reduced 29%-63% disease severity as effectively as the commercial product PL251 on Chinese cabbage in greenhouse trails. The in vitro egg infection rate was 47.83% by P. taiwanensis and 47.50% for Samsoniella sp., respectively. A special protocol for scanning electron microscope observation of the fungi-infected nematodes was established in this study, and the egg parasitism of the four fungi against M. enterolobii was further confirmed. For all fungi examined in this study, fungal hyphae were seen apparently penetrating into M. enterolobii eggs without destructive damage of the overall outer eggshell and the hyphae continued to grow within eggs after 6 days of infection. The results of this study imply a similar egg-parasitism mechanism for P. taiwanensis, Samsoniella sp., H. oviparasitica DoUCR50, and P. lilacinum PL251. It further enlightens the application potential of nematophagous fungi as biocontrol agents against plant-parasitic nematodes in vegetable crop management.

Control of Meloidogyne incognita in sweetpotato with fluensulfone.

In California, sweetpotato is mostly grown on light sandy soils in Merced County. Root-knot nematodes (Meloidogyne spp.) can reduce sweetpotato yields and quality. Fluensulfone is the active ingredient of the new non-fumigant nematicide Nimitz. Unlike fumigant nematicides, toxicity toward non-target organisms is low, and it does not emit volatile organic compounds which negatively impact air quality. In two field trials, the effect of fluensulfone on M. incognita levels, and on the yield and quality of sweetpotato was determined. Fluensulfone was applied as a pre-plant soil incorporated drench or as a drench followed by post-plant sprays. Fluensulfone treatments more than doubled the marketable yields over an untreated control and a metam-sodium treatment in both trials. It strongly reduced nematode symptoms on the harvested roots and nematode infestation of these roots. The lowest rate of fluensulfone was as effective as the higher rates, and post-plant sprays following a pre-plant soil incorporated drench did not result in any additional benefits. Fluensulfone did not reduce soil nematode levels at harvest. It was concluded that a pre-plant incorporated fluensulfone drench at a rate of 1.96 kg/ha could provide a viable alternative for currently used nematicides to mitigate root-knot nematode damage in sweetpotato.In California, sweetpotato is mostly grown on light sandy soils in Merced County. Root-knot nematodes (Meloidogyne spp.) can reduce sweetpotato yields and quality. Fluensulfone is the active ingredient of the new non-fumigant nematicide Nimitz. Unlike fumigant nematicides, toxicity toward non-target organisms is low, and it does not emit volatile organic compounds which negatively impact air quality. In two field trials, the effect of fluensulfone on M. incognita levels, and on the yield and quality of sweetpotato was determined. Fluensulfone was applied as a pre-plant soil incorporated drench or as a drench followed by post-plant sprays. Fluensulfone treatments more than doubled the marketable yields over an untreated control and a metam-sodium treatment in both trials. It strongly reduced nematode symptoms on the harvested roots and nematode infestation of these roots. The lowest rate of fluensulfone was as effective as the higher rates, and post-plant sprays following a pre-plant soil incorporated drench did not result in any additional benefits. Fluensulfone did not reduce soil nematode levels at harvest. It was concluded that a pre-plant incorporated fluensulfone drench at a rate of 1.96 kg/ha could provide a viable alternative for currently used nematicides to mitigate root-knot nematode damage in sweetpotato.

Effect of films on 1,3-dichloropropene and chloropicrin emission, soil concentration, and root-knot nematode control in a raised bed.

Soil fumigation is an important component of U.S. agriculture, but excessive emissions can be problematic. The objective of this study was to determine the effects of agricultural films (e.g., tarps) on soil fumigant atmospheric emissions and spatiotemporal distributions in soil, soil temperature, and plant pathogen control in the field using plastic films with various permeabilities and thermal properties. A reduced rate of 70% InLine (60.8% 1,3-dichloropropene (1,3-D) and 33.3% chloropicrin (CP)) was applied via drip line to raised soil beds covered with standard high-density polyethylene film (HDPE), thermic film (Thermic), or virtually impermeable film (VIF). 1,3-D and CP emission rates were determined using dynamic flux chambers, and the concentrations in soil were measured using a gas sampler. The pest control efficacy for the three treatments was determined using bioassay muslin bags containing soil infested with citrus nematodes (Tylenchulus semipenetrans). The results show that the Thermic treatment had the highest emission rates, followed by the HDPE and VIF treatments, and the soil concentrations followed the reverse order. In terms of pest control, covering the beds with thermic film led to sufficient and improved efficacy against citrus nematodes compared to standard HDPE film. Under HDPE, >20% of nematodes survived in the soil at 30 cm depth at day 12. The VIF treatment substantially reduced the emission loss from the bed (2% of the Thermic and 6% of the HDPE treatments) and eliminated plant parasitic nematodes because of its superior ability to entrap fumigant and heat within soils. The findings imply that not only the film permeability but also the synergistic ability to entrap heat should be considered in the development of new improved films for fumigation.

Correlations between root-associated microorganisms and peach replant disease symptoms in a California soil.

BACKGROUND: Replant disease often occurs when certain crops are "replanted" in a soil that had previously supported the same or similar plant species. This disease typically leads to reductions in plant growth, crop yields, and production duration, and its etiology remains ill-defined. The objective of this study was to identify microorganisms associated with peach replant disease symptoms at a field location in California, USA. Soil samples were subjected to treatments to create various levels of replant disease symptoms. Clonal peach seedlings were grown in the treated soils in greenhouse trials. After 6 weeks, plant growth parameters were measured, and both culture and culture-independent analyses were performed to identify root-associated bacteria, fungi and stramenopiles. RESULTS: A total of 295,785 bacterial operational taxonomic units (OTU) were identified by an Illumina-based, high throughput sequence analysis of rRNA genes. Among the 60 most abundant OTUs, 27 showed significant (P<0.05) negative correlation with peach shoot weights while 10 were positively correlated. Most of these OTUs belonged to the bacterial phylum Proteobacteria (96%), including the classes Gammaproteobacteria (44.4%), Betaproteobacteria (33.3%) and Alphaproteobacteria (22.2%), and the orders Pseudomonadales, Burkholderiales, Chromatiales, Rhodocyclales, and Sphingomonadales. The most abundant fungi were Trichoderma asperellum, Trichoderma virens, Fusarium oxysporum, Ceratocystis fimbriata and Fusarium solani. The most abundant stramenopiles were Pythium vexans, Pythium violae and an unidentified Aplanochytrium species. Validation experiments using sequence-selective quantitative PCR analyses identified negative and positive associations between P. vexans and Trichoderma spp. and peach shoot weights, respectively. CONCLUSIONS: This study identified numerous microorganisms associated with peach replant symptoms, some of which have been previously identified while others represent new candidates. Subsequent Koch's postulates investigations will assess their possible roles in this replant disease.

Biocontrol Efficacy Among Strains of Pochonia chlamydosporia Obtained from a Root-Knot Nematode Suppressive Soil.

Three Pochonia chlamydosporia var. chlamydosporia strains were isolated from a Meloidogyne incognita-suppressive soil, and then genetically characterized with multiple Pochonia-selective typing methods based on analysis of ß-tubulin, rRNA internal transcribed spacer (ITS), rRNA small subunit (SSU), and enterobacterial repetitive intergenic consensus (ERIC) PCR. All strains exhibited different patterns with the ERIC analysis. Strains 1 and 4 were similar with PCR analysis of ß-tubulin and ITS. The strains' potential as biological control agents against root-knot nematodes were examined in greenhouse trials. All three P. chlamydosporia strains significantly reduced the numbers of nematode egg masses. When chlamydospores were used as inoculum, strain 4 reduced egg numbers on tomato roots by almost 50%, and showed effects on the numbers of J2 and on nematode-caused root-galling. A newly developed SSU-based PCR analysis differentiated strain 4 from the others, and could therefore potentially be used as a screening tool for identifying other effective biocontrol strains of P. chlamydosporia var. chlamydosporia.

Assessment of Parasitic Activity of Fusarium Strains Obtained from a Heterodera schachtii-Suppressive Soil.

This study assessed the potential impact of various Fusarium strains on the population development of sugarbeet cyst nematodes. Fungi were isolated from cysts or eggs of Heterodera schachtii Schmidt that were obtained from a field suppressive to that nematode. Twenty-six strains of Fusarium spp. were subjected to a phylogenic analysis of their rRNA-ITS nucleotide sequences. Seven genetically distinct Fusarium strains were evaluated for their ability to influence population development of H. schachtii and crop performance in greenhouse trials. Swiss chard (Beta vulgaris) seedlings were transplanted into fumigated field soil amended with a single fungal strain at 1,000 propagules/g soil. One week later, the soil was infested with 250 H. schachtii J2/100 cm(3) soil. Parasitized eggs were present in all seven Fusarium treatments at 1,180 degree-days after fungal infestation. The percentage of parasitism ranged from 17 to 34%. Although the most efficacious F. oxysporum strain 471 produced as many parasitized eggs as occurred in the original suppressive soil, none of the Fusarium strains reduced the population density of H. schachtii compared to the conducive check. This supports prior results that Fusarium spp. were not the primary cause of the population suppression of sugarbeet cyst nematodes at this location.

Identifying microorganisms involved in specific pathogen suppression in soil.

Suppressive soils hold considerable potential for managing soilborne pathogens. When the suppressiveness has a biological origin, identifying the causal organisms is the crucial step in realizing this potential. Armed with such knowledge, it may be possible to develop effective and sustainable pest management strategies through application of these organisms or agronomic practices that influence their population densities. This chapter focuses on the development and utilization of a population-based approach for identifying microorganisms involved in specific pathogen suppression. Key experimental design principles of the approach are explored by examining experiments characterizing the biological nature of take-all decline. We also describe how this approach was used to identify microorganisms that suppress the sugarbeet cyst nematode. Additional experimental design considerations and future directions for such investigations are also discussed.

Induction of Beet-Cyst Nematode Suppressiveness by the Fungi Dactylella oviparasitica and Fusarium oxysporum in Field Microplots.

ABSTRACT The ability of Dactylella oviparasitica and Fusarium oxysporum to suppress Heterodera schachtii numbers was examined in field microplots. Fungi were individually added to fumigated field soil that was seeded with sugar beet. Four weeks later, soils were infested with H. schachtii second-stage juveniles (J2). At two harvests, 11 weeks and 19 weeks (1,469 and 2,547 degree days (base 8 degrees C), respectively) after nematode-infestation, H. schachtii cyst and egg numbers were assessed. At both time points, D. oviparasitica reduced H. schachtii population densities to those in the naturally suppressive soil, even when additional H. schachtii J2 were added to the microplots after the first harvest. Although F. oxy-sporum did not alter H. schachtii population densities after 11 weeks, significant reductions were detected after 19 weeks. The sustainability of the H. schachtii suppressiveness created by single applications of the fungi at the beginning of the microplot trials was further examined in a greenhouse study. Soil collected at the completion of the microplot trials was potted and seeded with sugar beet. Four weeks later, each pot was infested with H. schachtii J2. Approximately 16 weeks (1,389 degree days) after seeding, the D. oviparasitica-amended soil produced greater fresh root weights and considerably smaller nematode population densities than the nonamended control.

Suppression of the Plant-Parasitic Nematode Heterodera schachtii by the Fungus Dactylella oviparasitica.

This study examined the role of the fungi Dactylella oviparasitica and Fusarium oxysporum in the beet-cyst nematode (Heterodera schachtii) suppressiveness exhibited by a southern Californian soil. In prior research, the abundance of D. oviparasitica rRNA genes positively correlated with high levels of suppressiveness, whereas the abundance of F. oxysporum rRNA genes positively correlated with minimal to moderate levels of suppressiveness. In this report, both fungi were added to fumigation-induced nonsuppressive soil, planted with Swiss chard, and infested with H. schachtii juveniles. After two nematode generations, D. oviparasitica strain 50 reduced the population densities of H. schachtii eggs and juveniles to those in the suppressive soil and H. schachtii cysts to levels lower than in the suppressive soil. F. oxysporum did not significantly reduce H. schachtii populations. These results suggest that D. oviparasitica strain 50 plays a major role in the suppression of H. schachtii population development in this southern Californian soil.

Suppression of Heterodera schachtii Populations by Dactylella oviparasitica in Four Soils.

The effects of Dactylella oviparasitica strain 50 applications on sugarbeet cyst nematode (Heterodera schachtii) population densities and plant weights were assessed in four agricultural soils. The fungus was added to methyl iodide-fumigated and nonfumigated portions of each soil. The soils were seeded with Swiss chard. Four weeks later, soils were infested with H. schachtii second-stage juveniles (J2). Approximately 1,487 degree-days after infestation, H. schachtii cyst, egg and J2 numbers and plant weights were assessed. In all four fumigated soils, D. oviparasitica reduced all H. schachtii population densities and increased most of the plant weights compared to the nonamended control soils. In two of the nonfumigated soils (10 and SC), D. oviparasitica reduced H. schachtii population densities and increased most plant weight values compared to the nonamended control soils. For the other two nonfumigated soils (44 and 48), which exhibited pre-existing levels of H. schachtii suppressiveness, fungal applications had relatively little impact on H. schachtii population densities and plant weights. The results from this study combined with those from previous investigations suggest that D. oviparasitica strain 50 could be an effective biological control agent.

Degradation and adsorption of fosthiazate in soil.

Adsorption and degradation behavior of a pesticide in soil has a strong effect on its environmental fate as well as efficacy for pest control. Fosthiazate is an organophosphate compound that is currently under development as a nonfumigant nematicide. In this study, we evaluated adsorption and degradation kinetics of fosthiazate in three U.S. soils with different properties. Adsorption of fosthiazate in mineral soil was negligibly weak but appeared to increase with soil organic matter (OM) content. The half-life (T(1/2)) of fosthiazate ranged from 0.5 to 1.5 months in nonsterile soils but was prolonged to 1-3 months after sterilization. Degradation of fosthiazate in soil appeared to be caused by both chemical and microbial transformations. The persistence of fosthiazate generally decreased with increasing soil pH, but increased with increasing soil OM and clay contents. This results suggest that fosthiazate may have an enhanced leaching potential in acidic soils with low OM content, and its efficacy in high pH soils may not last as long as in neutral soils because of faster degradation.

Fumigant combinations for Cyperus esculentum L control.

The phase-out of methyl bromide as a soil fumigant has stimulated research into the use of other soil fumigants for weed control. Methyl bromide, methyl iodide, propargyl bromide, 1,3-dichloropropene (1,3-D) and metam-sodium were tested alone and in combination with chloropicrin in laboratory experiments to determine their efficacy against Cyperus esculentus L (yellow nutsedge) tubers. Propargyl bromide and metam-sodium were the most efficacious fumigants tested, with EC50 values of 3.7 and 6.5 microM, respectively. The relative potencies of methyl iodide and chloropicrin were not significantly different but were 2.6 and 2.9 times more potent than methyl bromide, respectively. The EC50 values for all fumigants other than 1,3-D were significantly lower than that of methyl bromide. Combining each fumigant with 17% chloropicrin resulted in a synergistic interaction. The greatest increase in potency between the expected result and the actual result was a relative potency of 3.8 with the methyl bromide/chloropicrin combination. The smallest increase in efficacy was with propargyl bromide and chloropicrin, with a relative potency of 1.5. There was no significant difference between the EC50 values of methyl bromide/chloropicrin and methyl iodide/chloropicrin combinations. Combining 1,3-D with 17% chloropicrin resulted in an EC50 value for C. esculentus control similar to that of methyl iodide applied alone.

Bacterial rRNA genes associated with soil suppressiveness against the plant-parasitic nematode Heterodera schachtii.

The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, alpha-Proteobacteria, beta-Proteobacteria, and gamma-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured alpha-proteobacterial clones was consistently associated with the highly suppressive treatments. A quantitative PCR analysis confirmed the association of this Rhizobium-like rDNA group with the H. schachtii suppressiveness.

Identification of Fungal rDNA Associated with Soil Suppressiveness Against Heterodera schachtii Using Oligonucleotide Fingerprinting.

ABSTRACT To understand the nature of a soil with suppressiveness against Heterodera schachtii, an rDNA analysis was used to identify fungi associated with H. schachtii cysts obtained from soils possessing various levels of suppressiveness. Because H. schachtii cysts isolated from these suppressive soils can transfer this beneficial property to nonsuppressive soils, analysis of the microorganisms associated with the cysts should lead to the identification of the causal organisms. Five soil treatments, generated by mixing different amounts of suppressive and fumigation-induced nonsuppressive soils, were infested with second-stage juveniles of H. schachtii and cropped with mustard-greens. Fungi were identified through an rDNA analysis termed oligonucleotide fingerprinting of ribosomal RNA genes (OFRG). Cysts obtained from soil mixtures consisting of 10 and 100% suppressive soil predominantly contained fungal rDNA with high sequence identity to Dactylella oviparasitica. The dominant fungal rDNA in the cysts isolated from the soil mixtures composed of 0.1 and 1% suppressive soil had high sequence identity to Fusarium oxysporum. Polymerase chain reaction (PCR) amplifications performed with sequence-selective primers corroborated the treatment-specific distribution of rDNA clones obtained by the OFRG analysis. When these sequence-selective PCR primers were used to examine H. schachtii cysts from biocidal soil treatments that produced various levels of suppressiveness, only the D. oviparasitica-like rDNA was consistently identified in the highly suppressive soils.

Population Dynamics of the Sting Nematode in California Turfgrass.

Population densities of Belonolaimus longicaudatus were monitored at monthly intervals at the Tamarisk country club golf course (1993 to 1994) and at the Annenburg Estates and Desert Island golf courses (1998). All three courses are located at Rancho Mirage, Coachella Valley, CA. The bermuda grass in the sampling area typically exhibited chlorosis at the beginning of April when the sting nematode populations began to increase. At the Tamarisk golf course, population density peaked in October, with 1,000 nematodes per 100 cm3 of soil, but declined rapidly, with the lowest population density occurring in December with approximately 50 nematodes per 100 cm3 of soil. At the Annenburg Estates and Desert Island golf courses, the nematode population densities peaked in June and July but declined rapidly to less than half of that density, presumably because of B. longicaudatus-caused host decline. Soil temperature and fluctuation of nematode densities were significantly correlated at all locations. Nematode distribution was greatest in the top 15 cm of soil except during the hottest summer months, when the population was higher at depths of 15 to 30 cm.