A Spatiotemporal Analysis and Dispersal Patterns of the Potato Cyst Nematode Globodera pallida in Idaho.

The potato cyst nematode Globodera pallida is a globally regulated potato pest. It was detected for the first time in the United States in the state of Idaho in 2006, and as of February 2019, the infestation is limited to 1,326 hectares. G. pallida is a specialized obligate sedentary endoparasite that can survive in the soil for up to 30 years in the absence of its potato host. In highly infested fields, the nematode can reduce tuber yields up to 80% and is spread mainly through the movement of soil, tubers, or farm equipment. The objectives of this study were to describe the spatiotemporal pattern of G. pallida in infested fields and model its dispersal patterns in southeastern Idaho. We used geostatistical tools and simulation models for precise mapping and to describe the relationships between G. pallida incidence and the spatial configurations. We found that the nematode is spatially clustered and prevalent around edges of fields, and its dispersal pattern followed the direction of cultivation. We found that the absence of potato in an infested field significantly reduced the number of cysts sampled each year subsequent to the initial delimitation sampling in 2007. Phytosanitary measures prohibiting the growth of potato contributed to stopping nematode reproduction, and the use of chemical fumigants and biofumigant cover crops contributed to a significant reduction in egg viability. We observed a process of a nonlinear decline in the prevalence of cysts as the distance separation from the primary infestation focus increased. A power law model was used to fit G. pallida dispersal capabilities. This study contributed to describing G. pallida infestation for Idaho. The goal of this study is to provide information on the spatial pattern and landscape ecology of G. pallida in Idaho for policy makers, industry, and researchers as well as facilitate common understandings on the challenges and opportunities for controlling this pest in Idaho.

A Predictive Risk Model Analysis of the Potato Cyst Nematode Globodera pallida in Idaho.

Globodera pallida is a major nematode pest of potato (Solanum tuberosum) and is of great economic importance for the potato industry. Assessing potato yield loss caused by the Idaho G. pallida population under field conditions was not performed due to its quarantine status in Idaho, where it is prohibited by regulatory statutes to grow potato in any infested fields. The experimental data came from three trials that were conducted under greenhouse conditions. A predictive risk model analysis was performed to: (i) determine the effect of the Idaho population of G. pallida on potato yield; (ii) estimate reproduction rate from different initial nematode densities; and (iii) simulate potato yield losses in Idaho field conditions by integrating the coefficients of potato yield into the SUBSTOR-DSSAT crop simulation model. Experiments were conducted under greenhouse conditions using five initial G. pallida soil infestation levels (0, 10, 20, 40, and 80 eggs/g soil). The coefficients of potato yield achieved under each initial nematode density were integrated into the SUBSTOR-DSSAT potato growth simulation model. The model showed that tuber weight reached a maximum yield of 96 ton/ha in noninfested soil. Based on the greenhouse trials, the model predicted a minimum yield of 12 and 58 ton/ha in trial 1 and trial 2/3 respectively, when initial nematode density was 80 eggs/g soil. In trial 1, tuber weight was significantly reduced by 44% at 40 eggs/g soil and by 87% at 80 eggs/g soil, and 20% at 40 eggs/g soil and by 39% at 80 eggs/g soil in trial 2/3. The outputs of this study should facilitate common understanding between regulators, policymakers, and potato growers on the challenges and opportunities for controlling this economically important pest in Idaho.

A Spatial Analysis of the Potato Cyst Nematode Globodera pallida in Idaho.

The potato cyst nematode Globodera pallida is a globally regulated and quarantine potato pest. It was detected for the first time in the United States in the state of Idaho in 2006. A spatial analysis was performed to (i) understand the spatial arrangement of fields infested with G. pallida in southern Idaho using spatial point pattern analysis, and (ii) evaluate the potential threat of G. pallida for entry to new areas using spatial interpolation techniques. Data point locations, cyst numbers and egg viability values for each infested field were collected by USDA-APHIS during 2006 to 2014. Results showed the presence of spatially clustered fields infested with G. pallida (P = 0.003). We determined that the spread of G. pallida grew in diameter from the original center of infestation toward the southwest as an ellipsoidal-shaped cluster. Based on the aggregated spatial pattern of distribution, we determined that G. pallida spread followed a contagion effect scenario, where nearby infested fields contributed to the infestation of new fields, probably through soil contaminated agricultural equipment or tubers. We determined that the presence of G. pallida in southern Idaho is unlikely to be associated with new introductions from outside the state of Idaho. The aggregation pattern of fields infested with G. pallida, with an average of 4,263 cysts/ha and egg viability of 25%, facilitates quarantine activities and confines the propagation of this pest to a small area, which in 2017 was estimated to be 1,233 ha. The tools and methods provided in this study facilitate comprehensive approaches to improve G. pallida control and eradication programs as well as to raise public awareness of the problems surrounding this economically important potato pest.

Individual-based approach to modeling hyphal growth of a biocontrol fungus in soil.

ABSTRACT Fungi in soil perform beneficial roles that include biological control of soilborne plant pathogens. However, relatively little predictive information is available about the growth and activity of fungal hyphae in soil habitats. A stochastic computer simulation model ("Fungmod") was developed to predict hyphal growth of the biocontrol fungus Trichoderma harzianum ThzID1 in soil. The model simulates a fungal colony as a population of spatially referenced hyphal segments, and is individual-based, in that records of spatial location and branching hierarchy are maintained for individual hyphal nodes. In this way, the entire spatial structure of the fungal colony (hyphal network) can be explicitly reconstructed at any point in time. Also, the soil habitat is modeled as a population of spatially referenced 1-mm(3) soil cells, allowing for the simulation of a spatially heterogeneous environment. Initial hyphal growth parameters were derived from previously published results, and the model was tested against new data derived from image analysis of hyphal biomass accumulation in soil. The ability to predict fungal growth in natural habitats will help to improve the predictability of successful myco-parasitic events in biological control systems.

Use of Green Fluorescent Protein and Image Analysis to Quantify Proliferation of Trichoderma harzianum in Nonsterile Soil.

ABSTRACT One drawback of traditional methods for fungal biomass measurement is the inability to distinguish biomass of an introduced fungus from that of the indigenous microbial community in nonsterile soil. We quantified biomass of a specific fungal biological control agent in nonsterile soil using epifluorescence microscopy and image analysis of green fluorescent protein (GFP)-expressing Trichoderma harzianum (ThzID1-M3). Numbers of colony forming units on a semiselective medium were compared with biomass estimates from image analysis, after ThzID1-M3 was incubated in soil that either remained moist (-0.05 MPa) for 14 to 21 days or remained moist for approximately 5 days and then was allowed to dry to <-3.0 MPa. Recovery of significant numbers of ThzID1-M3 propagules lagged approximately 3 days behind initiation of hyphal growth. Reductions in both colony counts and biomass were observed over time when soil was allowed to dry. However, in soil that remained moist, colony counts increased over a 14- to 21-day period even though biomass declined after approximately 3 to 5 days. Our results confirm that use of GFP, along with epifluorescence microscopy, is a useful tool to distinguish active hyphal biomass, the form of the fungus that is functional for biological control, from inactive propagules such as conidia or chlamydospores that are enumerated by plate counts.

Isothiocyanates Produced by Brassicaceae Species as Inhibitors of Fusarium oxysporum.

Glucosinolates contained in members of the Brassicaceae release isothiocyanates potentially useful in controlling Fusarium oxysporum pathogens in conifer seedling nursery soils. Our objective was to determine the toxicity of individual isothiocyanates to different growth stages of the fungus. Bioassays with four F. oxysporum isolates were conducted using sealed containers in which 0.3 µl of 2-propenyl, ethyl, buty, phenylethyl, benzyl, or phenyl isothiocyanate was allowed to volatilize. Propenyl and ethyl isothiocyanates were the most fungistatic of those compounds tested. The same concentrations of propenyl and ethyl isothiocyanates that inhibited mycelial growth completely suppressed conidial and chlamydospore germination of all isolates. Other isothiocyanates including ethyl, benzyl, and phenethyl were also fungitoxic to F. oxysporum conidia and chlamydospores. Reduction in pathogen populations resulting from a green-manure crop are likely achievable since chlamydospores are sensitive to isothiocyanate. Pathogenic F. oxysporum isolates infesting nursery soils would likely be most suppressed by species of plants such as Brassica carinata, B. nigra, and B. juncea, which contain glucosi-nolates that release high concentrations of propenyl isothiocyanate.

Influence of a Fungus-Feeding Nematode on Growth and Biocontrol Efficacy of Trichoderma harzianum.

ABSTRACT A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80 degrees C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.

Combined effects of Brassica napus seed meal and Trichoderma harzianum on two soilborne plant pathogens.

The effects of soil amendment with rapeseed meal from Brassica napus cv. 'Dwarf Essex' (high glucosinolate concentrations) and 'Stonewall' (low glucosinolate concentrations) on the biological control activity of Trichoderma harzianum towards Sclerotinia sclerotiorum and Aphanomyces euteiches were evaluated. Trichoderma harzianum added to soil reduced myceliogenic germination of S. sclerotiorum by 94%, but did not affect carpogenic germination. In contrast, 100% reduction in carpogenic germination was observed in soil amended with Dwarf Essex meal, along with a 33% reduction in myceliogenic germination. With Stonewall meal as soil amendment, carpogenic germination was reduced by 44% and myceliogenic germination was not affected. Both Dwarf Essex and Stonewall meals inhibited colonization of sclerotia in soil by T. harzianum, from 100% to 0% and 8%, respectively, so that biocontrol activity of T. harzianum was reduced in the presence of either meal. Aphanomyces euteiches root rot of pea was significantly reduced by T. harzianum alone (100%), by amendment with Dwarf Essex meal alone (77%), and by T. harzianum in combination with Dwarf Essex meal (100%). Amendment with Stonewall meal alone did not control root rot, and combination of Stonewall meal with T. harzianum reduced the biocontrol efficacy of T. harzianum.

Cotransformation of Trichoderma harzianum with beta-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils.

Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), beta-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.

Spatial Patterns of Rhizoplane Populations of Pseudomonas fluorescens.

Geostatistical analysis was used to compare rhizoplane colonization patterns of an antibiotic-producing biological control bacterium versus a non-antibiotic-producing mutant strain. Pea seeds were inoculated with Pseudomonas fluorescens 2-79RN(inf10) or P. fluorescens 2-79-B46 (a phenazine-deficient Tn5 mutant of P. fluorescens 2-79RN(inf10)) (10(sup8) CFU/pea), planted in sterile sand, and incubated at 20(deg)C. After 3 days, seedlings were prepared for scanning electron microscopy. Photomicrographs (x1,000) of the root surface were taken at the seed-root junction and at 0.5-cm intervals to the root tip. Bacterial counts on the root surface were made in 5- by 5-(mu)m sample units over an area which was 105 by 80 (mu)m. Coordinates and number of bacteria were recorded for each sample unit. Spatial statistics were calculated by covariance for the following directions: omnidirectional, 0, 45, 90, and 135(deg). The ranges of spatial influence and nugget (estimator of spatially dependent variation) were determined. For both P. fluorescens 2-79RN(inf10) and P. fluorescens 2-79-B46, spatial structure was evident along the entire root, particularly in the 0(deg) direction (along the root length) (e.g., range = 24 (mu)m, nugget = 0.52). The degree of spatial dependence observed indicated aggregation of bacterial cells. No differences were detected in the spatial patterns of colonies of P. fluorescens 2-79RN(inf10) and P. fluorescens 2-79-B46, indicating that the lack of phenazine production did not influence spatial patterns on the rhizoplane.

Toxicity of Glucosinolate Degradation Products from Brassica napus Seed Meal Toward Aphanomyces euteiches f. sp. pisi.

ABSTRACT Brassica tissues are potentially useful in the control of Aphanomyces root rot of peas (Pisum sativum), but identity of the responsible compounds and specific impacts of those compounds on the pathogen's infection potential remain uncertain. Brassica napus seed meals and water extracts from these meals were used to determine the effect of glucosinolate hydrolysis products on Aphanomyces euteiches f. sp. pisi. B. napus meal ('Dwarf Essex') containing glucosinolates and intact myrosinase, the enzyme responsible for glucosinolate hydrolysis, completely inhibited infection by A. euteiches f. sp. pisi oospores. Water extracts from this meal, likewise, severely inhibited infection by oospores, as well as mycelial growth. Extracts from autoclaved 'Dwarf Essex' meal, in which myrosinase was denatured, and a low glucosinolate B. napus variety ('Stonewall') produced little disease reduction and had less impact on mycelial growth. Gas chromatographic analysis of Brassica tissues and water extracts confirmed that glucosinolates remained in autoclaved 'Dwarf Essex' meal and that 'Stonewall' meal contained low glucosinolate concentrations. 5-Vinyloxazolidine-2-thione was identified by mass spectrometry as a dominant glucosinolate hydrolysis product in aqueous extracts of the inhibitory meal. Bioassays conducted with aqueous solutions of this compound reduced mycelial growth, but not to the extent of those from intact 'Dwarf Essex' meal. Water-soluble compounds produced from the hydrolysis of glucosinolates in B. napus tissues reduced A. euteiches oospore infection and inhibited mycelial growth, thus, demonstrating potential utility of Brassica species in the control of A. euteiches.

Inhibition of Aphanomyces euteiches f. sp. pisi by Volatiles Produced by Hydrolysis of Brassica napus Seed Meal.

Seed meal from Brassica napus (rapeseed) produced volatile fungitoxic compounds potentially of value in the control of Aphanomyces root rot of pea. Hyphal growth, germination of encysted zoospores, and oospore survival and inoculum potential, were determined in the presence of volatiles produced from B. napus seed meal. Volatile compounds from B. napus meal completely suppressed mycelial growth and germination of encysted zoospores on agar. In growth chamber bioassays, pea (Pisum sativum) seed inoculated with zoospore suspensions and incubated 24 h in the presence of volatiles from rapeseed meal had 50% lower root rot disease severity than in the absence of meal. Volatile compounds passing through soil also significantly decreased survival and inoculum potential of oospores. Gas chromatographic analysis of rapeseed tissues and the volatile compounds evolved from tissues showed that substrate glucosinolates were hydrolyzed enzymatically to produce mainly isothiocyanates. Non-autoclaved rapeseed meal produced significantly higher levels of volatile compounds than did autoclaved meal. Also, volatile compounds produced from autoclaved meal were dominated by nitriles, whereas isothiocyanates were more common volatile products from non-autoclaved meal. Our results indicate that B. napus allelochemicals responsible for toxic effects toward A. euteiches f. sp. pisi are enzymatic hydrolysis products of glucosinolates.

Effect of parental growth on dynamics of conjugative plasmid transfer in the pea spermosphere.

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.

Method to enhance growth and sporulation of pelletized biocontrol fungi.

The biocontrol fungi Trichoderma harzianum, used to control soilborne plant pathogens, and Beauveria bassiana, used to control insect pests, were formulated as mycelial biomass in alginate pellets with wheat bran added. After drying for 0, 4, or 16 h, pellets were placed in water or in aqueous solutions of polyethylene glycol (PEG) 8000 for 4 to 24 h and then allowed to continue drying. PEG-treated pellets containing T. harzianum showed significantly greater proliferation of hyphae in soil than untreated pellets or pellets treated with water. Production of conidia of T. harzianum from PEG-treated pellets was lower than production from untreated pellets after 4 days, although rates were equivalent after 7 days. In contrast, production of conidia of B. bassiana was significantly more rapid from PEG-treated pellets than from untreated pellets. Biocontrol of soilborne plant pathogens or insect pests may be enhanced by rapid hyphal growth of T. harzianum in soil or rapid sporulation of B. bassiana on foliage, respectively. Therefore, PEG treatment may improve the efficacy of these biocontrol agents.

Model to predict aerial dispersal of bacteria during environmental release.

Risk assessment for genetically engineered bacteria sprayed onto crops includes determination of off-site dispersal and deposition. The ability to predict microbial dispersal patterns is essential to characterize the uncertainty (risk) associated with environmental release of recombinant organisms. Toward this end, a particle dispersal model was developed to predict recovery of bacteria on fallout plates at various distances and directions about a test site. The microcomputer simulation incorporates particle size distribution, wind speed and direction, turbulence, evaporation, sedimentation, and mortality, with a time step of 0.5 s. The model was tested against data reported from three field applications of nonrecombinant bacteria and two applications of recombinant bacteria. Simulated dispersal of 10(5) particles was compared with reported deposition measurements. The model may be useful in defining appropriate populations of organisms for release, methods of release or application, characteristics of a release site that influence containment or dispersal, and in developing an appropriate sampling methodology for monitoring the dispersal of organisms such as genetically engineered bacteria.

Aerial Dispersal and Epiphytic Survival of Pseudomonas syringae during a Pretest for the Release of Genetically Engineered Strains into the Environment.

Prospective experimental field evaluation of genetically engineered microorganisms, such as microbial pest control agents, raises issues of how to properly ascertain their fate and survival in the environment. Field trials with recombinant organisms must reflect requirements for sampling and monitoring. Field trials were conducted at Tulelake, Calif., to monitor the numbers of viable cells of a nonrecombinant strain of Pseudomonas syringae that entered the atmosphere and landed on plants and soil during and after an aerosol spray application. An exponential decrease in numbers of viable cells deposited at increasing distances from three sprayed plots was observed. The relative rate of survival of cells sprayed directly on plants was more than 10 times higher than that of cells dispersed through the air to similar adjacent plants. Results are being used to gain experience with the characteristics of a release site that influence containment or dispersal and to develop appropriate sampling methodologies for evaluating survival and dispersal characteristics of genetically engineered bacteria released into the environment. The ability to make predictions about microbial dispersal and survival will reduce the uncertainties associated with environmental releases of recombinant organisms.

Predictive model of conjugative plasmid transfer in the rhizosphere and phyllosphere.

A computer simulation model was used to predict the dynamics of survival and conjugation of Pseudomonas cepacia (carrying the transmissible recombinant plasmid R388:Tn1721) with a nonrecombinant recipient strain in simple rhizosphere and phyllosphere microcosms. Plasmid transfer rates were derived for a mass action model, and donor and recipient survival were modeled as exponential growth and decay processes or both. Rate parameters were derived from laboratory studies in which donor and recipient strains were incubated in test tubes with a peat-vermiculite solution or on excised radish or bean leaves in petri dishes. The model predicted donor, recipient, and transconjugant populations in hourly time steps. It was tested in a microcosm planted with radish seeds and inoculated with donor and recipient strains and on leaf surfaces of radish and bean plants also growing in microcosms. Bacteria were periodically enumerated on selective media over 7 to 14 days. When donor and recipient populations were 10(6) to 10(8) CFU/g (wet weight) of plant or soil, transconjugant populations of about 10(1) to 10(4) were observed after 1 day. An initial rapid increase and a subsequent decline in numbers of transconjugants in the rhizosphere and on leaf surfaces were correctly predicted.