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1.  Specific Microbial Attachment to Root Knot Nematodes in Suppressive Soil 
Understanding the interactions of plant-parasitic nematodes with antagonistic soil microbes could provide opportunities for novel crop protection strategies. Three arable soils were investigated for their suppressiveness against the root knot nematode Meloidogyne hapla. For all three soils, M. hapla developed significantly fewer galls, egg masses, and eggs on tomato plants in unsterilized than in sterilized infested soil. Egg numbers were reduced by up to 93%. This suggested suppression by soil microbial communities. The soils significantly differed in the composition of microbial communities and in the suppressiveness to M. hapla. To identify microorganisms interacting with M. hapla in soil, second-stage juveniles (J2) baited in the test soil were cultivation independently analyzed for attached microbes. PCR-denaturing gradient gel electrophoresis of fungal ITS or 16S rRNA genes of bacteria and bacterial groups from nematode and soil samples was performed, and DNA sequences from J2-associated bands were determined. The fingerprints showed many species that were abundant on J2 but not in the surrounding soil, especially in fungal profiles. Fungi associated with J2 from all three soils were related to the genera Davidiella and Rhizophydium, while the genera Eurotium, Ganoderma, and Cylindrocarpon were specific for the most suppressive soil. Among the 20 highly abundant operational taxonomic units of bacteria specific for J2 in suppressive soil, six were closely related to infectious species such as Shigella spp., whereas the most abundant were Malikia spinosa and Rothia amarae, as determined by 16S rRNA amplicon pyrosequencing. In conclusion, a diverse microflora specifically adhered to J2 of M. hapla in soil and presumably affected female fecundity.
doi:10.1128/AEM.03905-13
PMCID: PMC3993313  PMID: 24532076
2.  Contributions of Fusarium virguliforme and Heterodera glycines to the Disease Complex of Sudden Death Syndrome of Soybean 
PLoS ONE  2014;9(6):e99529.
Background
Sudden death syndrome (SDS) of soybean caused by Fusarium virguliforme spreads and reduces soybean yields through the North Central region of the U.S. The fungal pathogen and Heterodera glycines are difficult to manage.
Methodology/Principal Findings
The objective was to determine the contributions of H. glycines and F. virguliforme to SDS severity and effects on soybean yield. To quantify DNA of F. virguliforme in soybean roots and soil, a specific real time qPCR assay was developed. The assay was used on materials from soybean field microplots that contained in a four-factor factorial-design: (i) untreated or methyl bromide-fumigated; (ii) non-infested or infested with F. virguliforme; (iii) non-infested or infested with H. glycines; (iv) natural precipitation or additional weekly watering. In years 2 and 3 of the trial, soil and watering treatments were maintained. Roots of soybean ‘Williams 82’ were collected for necrosis ratings at the full seed growth stage R6. Foliar symptoms of SDS (area under the disease progress curve, AUDPC), root necrosis, and seed yield parameters were related to population densities of H. glycines and the relative DNA concentrations of F. virguliforme in the roots and soil. The specific and sensitive real time qPCR was used. Data from microplots were introduced into models of AUDPC, root necrosis, and seed yield parameters with the frequency of H. glycines and F. virguliforme, and among each other. The models confirmed the close interrelationship of H. glycines with the development of SDS, and allowed for predictions of disease risk based on populations of these two pathogens in soil.
Conclusions/Significance
The results modeled the synergistic interaction between H. glycines and F. virguliforme quantitatively in previously infested field plots and explained previous findings of their interaction. Under these conditions, F. virguliforme was mildly aggressive and depended on infection of H. glycines to cause highly severe SDS.
doi:10.1371/journal.pone.0099529
PMCID: PMC4059700  PMID: 24932970
3.  Predicting Damage of Meloidogyne incognita on Watermelon 
Journal of Nematology  2012;44(2):127-133.
Quantitative growth response of watermelon (Citrullus lanatus) sensitive to Meloidogyne incognita is poorly understood. Determination of soil population densities of second-stage juveniles (J2) of M. incognita with Baermann funnel extraction often is inaccurate at low soil temperatures. In greenhouse experiments, three sandy soils were inoculated with dilution series of population densities of eggs or J2 of M. incognita and planted in small containers to watermelon ‘Royal Sweet’ or subjected to Baermann funnel extraction. After five weeks of incubation in the greenhouse bioassay plants in egg-inoculated soils, gall numbers on watermelon roots related more closely to inoculated population densities than J2 counts after Baermann funnel extraction. In April 2004, perpendicularly-inserted tubes (45-cm diameter, 55-cm deep) served as microplots where two methyl bromide-fumigated sandy soils were inoculated with egg suspensions of M. incognita at 0, 100, 1,000 or 10,000 eggs/100 cm3 of soil in 15-cm depth. At transplanting of 4-week old watermelon seedlings, soils were sampled for the bioassay or for extraction of J2 by Baermann funnel. In the Seinhorst function of harvested biomass in relation to nematode numbers, decline of biomass with increasing population densities of M. incognita was accurately modeled by the inoculated eggs (R2 = 0.93) and by the counts of galls on the bioassay roots (R2 = 0.98); but poorly by J2 counts (R2 = 0.68). Threshold levels of watermelon top dry weight to M. incognita were 122 eggs/100 cm3 soil, 1.6 galls on bioassay roots, or 3.6 J2/100 cm3 of soil. Using the bioassay in early spring for predicting risk of nematode damage appeared useful in integrated pest management systems of watermelon.
PMCID: PMC3578463  PMID: 23482631
bioassay; root-knot nematode; Seinhorst function; threshold level
4.  General Suppression of Escherichia coli O157:H7 in Sand-Based Dairy Livestock Bedding▿ †  
Sand bedding material is frequently used in dairy operations to reduce the occurrence of mastitis and enhance cow comfort. One objective of this work was to determine if sand-based bedding also supported the microbiologically based suppression of an introduced bacterial pathogen. Bedding samples were collected in summer, fall, and winter from various locations within a dairy operation and tested for their ability to suppress introduced populations of Escherichia coli O157:H7. All sources of bedding displayed a heat-sensitive suppressiveness to the pathogen. Differences in suppressiveness were also noted between different samples at room temperature. At just 1 day postinoculation (dpi), the recycled sand bedding catalyzed up to a 1,000-fold reduction in E. coli counts, typically 10-fold greater than the reduction achieved with other substrates, depending on the sampling date. All bedding substrates were able to reduce E. coli populations by over 10,000-fold within 7 to 15 dpi, regardless of sampling date. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to identify bacterial populations potentially associated with the noted suppression of E. coli O157:H7 in sand bedding. Eleven terminal restriction fragments (TRFs) were overrepresented in paired comparisons of suppressive and nonsuppressive specimens at multiple sampling points, indicating that they may represent environmentally stable populations of pathogen-suppressing bacteria. Cloning and sequencing of these TRFs indicated that they represent a diverse subset of bacteria, belonging to the Cytophaga-Flexibacter-Bacteroidetes, Gammaproteobacteria, and Firmicutes, only a few of which have previously been identified in livestock manure. Such data indicate that microbial suppression may be harnessed to develop new options for mitigating the risk and dispersal of zoonotic bacterial pathogens on dairy farms.
doi:10.1128/AEM.01655-10
PMCID: PMC3067323  PMID: 21257815
5.  Sustainable Approaches to the Management of Plant-parasitic Nematodes and Disease Complexes 
Journal of Nematology  2011;43(2):122-125.
Physical, chemical, and biological factors of soil may reduce damage caused by plant-parasitic nematodes. Suppression of plant-parasitic nematodes is particularly challenging in soils in which there are short crop sequences, sequential susceptible host crops, or infestations of multiple nematode species. In southern Indiana, a watermelon production system involving rotations with soybean and corn does not suppress Meloidogyne incognita, but several aspects of such systems can be modified to reduce nematode damage in an integrated management approach. Cash crops with resistance to M. incognita can be used to reduce population densities of M. incognita. Small grains as cover crops can be replaced by cover crops with resistance to M. incognita or by crops with biofumigation potential. Mycorrhizal fungal inoculations of potting mixes during transplanting production of watermelon seedlings may improve early crop establishment. Other approaches to nematode management utilize soil suppressiveness. One-year rotations of soybean with corn neither reduced the soil-borne complex of sudden death syndrome (SDS) nor improved soybean root health over that in soybean monoculture. Reduced tillage combined with crop rotation may reduce the activity of soil-borne pathogens in some soils. For example in a long-term trial, numbers of Heterodera glycines and severity of foliar SDS symptoms were reduced under minimum tillage. Thus, sustainable management strategies require holistic approaches that consider entire production systems rather than focus on a single crop in its year of production.
PMCID: PMC3380463  PMID: 22791923
6.  Detection of Suppressiveness against Rotylenchulus reniformis in Soil from Cotton (Gossypium hirsutum) Fields in Texas and Louisiana 
Journal of nematology  2008;40(1):35-38.
Rotylenchulus reniformis is a major problem confronting cotton production in the central part of the cotton belt of the United States of America. In this study, the hypothesis that natural antagonists in some cases are responsible for unusually low densities of the nematode in certain fields was tested by assaying soils from 22 selected fields for the presence of transferable agents in pots containing cotton plants. In one field, soil from four different depth ranges was tested. In the first of two types of assays, 1 part nematode infested soil was added to 9 parts test soil that was left untreated or autoclaved before mixing; this mixture was used to fill pots. In the second type of assay, 1 part test soil was added to 9 or 19 parts pasteurized fine sand, and nematodes were introduced in aqueous suspension. In three experiments representing both types of assay, transferable or autoclavable agent(s) from four fields in South Texas suppressed nematode populations by 48, 78, 90 and 95%. In one experiment, transferable agents in five fields in Louisiana suppressed populations from 37 to 66%. Identification and evaluation of these agents for biological control of R. reniformis merits further study.
PMCID: PMC2586520  PMID: 19259517
biological control; cotton; Gossypium hirsutum; Rotylenchulus reniformis; reniform nematode; soil suppressiveness
7.  Detection and Description of Soils with Specific Nematode Suppressiveness 
Journal of Nematology  2005;37(1):121-132.
Soils with specific suppressiveness to plant-parasitic nematodes are of interest to define the mechanisms that regulate population density. Suppressive soils prevent nematodes from establishing and from causing disease, and they diminish disease severity after initial nematode damage in continuous culturing of a host. A range of non-specific and specific soil treatments, followed by infestation with a target nematode, have been employed to identify nematode-suppressive soils. Biocidal treatments, soil transfer tests, and baiting approaches together with observations of the plant-parasitic nematode in the root zone of susceptible host plants have improved the understanding of nematode-suppressive soils. Techniques to demonstrate specific soil suppressiveness against plant-parasitic nematodes are compared in this review. The overlap of studies on soil suppressiveness with recent advances in soil health and quality is briefly discussed. The emphasis is on methods (or criteria) used to detect and identify soils that maintain specific soil suppressiveness to plant-parasitic nematodes. While biocidal treatments can detect general and specific soil suppressiveness, soil transfer studies, by definition, apply only to specific soil suppressiveness. Finally, potential strategies to exploit suppressive soils are presented.
PMCID: PMC2620936  PMID: 19262851
biological control; cyst nematodes; cyst nematode-suppressive soil; density dependence; heat treatments; Heteordera avenae; H. glycines; H. schachtii

Results 1-7 (7)