Extraction of Cyst Nematodes from Organic Soils

The effects of extraction technique, sample size, soil moisture level, and overflow rate on recovery of Globodera rostochiensis and (or) Heterodera schachtii cysts from organic soils were investigated. A modified Fenwick can (MFC) and an underflow elutriator (UE) described in this paper were evaluated and compared for cyst recovery efficiency and amount of organic flotsam collected. The MFC and UE extracted similar numbers of cysts, but the UE collected 50% less flotsam than the MFC. Sample size was negatively correlated with cyst recovery and positively correlated with amount of flotsam. The amount of flotsam recovered with the MFC was correlated with overflow speed. Presoaking air dried samples for 30 minutes halved the amount of flotsam without affecting cyst recovery. Extracting cysts from wet soil without prior drying resulted in negligible recovery with both extraction techniques. There were no significant differences in cyst recovery of the two genera tested.     

Probability of Globodera rostochiensis Spread on Equipment and Potato Tubers

The probability of spreading cysts of Globodera rostochiensis on farming equipment and potato tubers was investigated in naturally infested field plots. The number of cysts recovered from soil that adhered to equipment differed significantly between different pieces of equipment. These differences were related to initial nematode density and, in most cases, to the volume of soil that adhered to the equipment. At an initial density of 0.04 egg/cm³ of soil, significantly more cysts were recovered from a potato digger than from a potato hiller, cultivator, or plow. At an initial density of 0.90 egg/cm³ of soil, significantly more cysts were recovered from the plow than from the other equipment. Although the population density was 22 times greater, only 10 times more cysts adhered 3 to equipment used in soil with a density of 0.90 egg/cm³ of soil than when used in soil infested at 0.04 egg/cm³. The number of potato tuber samples (4.5 kg) that contained cysts with viable eggs was positively correlated with the initial densities of G. rostochiensis in soil in which they were produced. The percentage of tuber samples with cysts containing viable eggs was 10-12% for tubers harvested from soil with densities less than 1 egg/cm³ and 30-76% for tubers harvested from soil with densities greater than 4 eggs/cm³ of soil.     

Effect of Compost and Manure Soil Amendments on Nematodes and on Yields of Potato and Barley: A 7-Year Study

A 7-year study located in Prince Edward Island, Canada, examined the influence of compost and manure on crop yield and nematode populations. The compost used in this study consisted of cull waste potatoes, sawdust, and beef manure in a 3:3:1 ratio, respectively. No plant-parasitic nematodes were detected in samples collected from windrow compost piles at 5- and 30-cm depths prior to application on field plots. Low population densities of bacterial-feeding nematodes were recovered from compost windrows at the 5-cm depth. Field plots of potato (Solanum tuberosum cv. Kennebec) received compost applied at 16 metric tonnes per hectare, or beef manure applied at 12 metric tonnes per hectare. An adjacent trial with barley (Hordeum vulgare cv. Mic Mac) received only the compost treatment. In both trials the experimental design was a complete randomized block with four replicates. Data averaged over seven growing seasons indicated that population levels of root-lesion nematodes (primarily Pratylenchus penetrans) were higher in root-zone soil in potato plots treated with either compost or manure compared to the untreated control plots. The soil amendments did not affect root-knot nematode (Meloidogyne hapla) population densities in the potato plots, but clover-cyst nematodes (Heterodera trifolii) were more numerous in the root-zone soils of barley treated with compost compared to the untreated plots. Numbers of bacterial-feeding nematodes (primarily Diplogaster lheritieri) were greater in soil in potato plots treated with manure and in soil around barley roots than in untreated plots. Total yields of potato tubers averaged over seven growing seasons increased by 27% in the plots treated with either compost or manure. Grain yields of barley also were increased by 12% when compost was applied. These results indicated that organic amendments increased crop yields, but the impacts on different nematode species varied and usually increased soil population levels.     

Soil Organic Matter and Management of Plant-Parasitic Nematodes

Organic matter and its replenishment has become a major component of soil health management programs. Many of the soil''s physical, chemical, and biological properties are a function of organic matter content and quality. Adding organic matter to soil influences diverse and important biological activities. The diversity and number of free-living and plant-parasitic nematodes are altered by rotational crops, cover crops, green manures, and other sources of organic matter. Soil management programs should include the use of the proper organic materials to improve soil chemical, physical, and biological parameters and to suppress plant-parasitic nematodes and soilborne pathogens. It is critical to monitor the effects of organic matter additions on activities of major and minor plant-parasitic nematodes in the production system. This paper presents a general review of information in the literature on the effects of crop rotation, cover crops, and green manures on nematodes and their damage to economic crops.     

Embracing the Emerging Precision Agriculture Technologies for Site-Specific Management of Yield-Limiting Factors

Precision agriculture (PA) is providing an information revolution using Global Positioning (GPS) and Geographic Information (GIS) systems and Remote Sensing (RS). These technologies allow better decision making in the management of crop yield-limiting biotic and abiotic factors and their interactions on a site-specific (SSM) basis in a wide range of production systems. Characterizing the nature of the problem(s) and public education are among the challenges that scientists, producers, and industry face when adapting PA technologies. To apply SSM, spatio-temporal characteristics of the problem(s) need to be determined and variations within a field demonstrated. Spatio-temporal characteristics of a given pathogen or pest problem may be known but may not be the only or primary cause of the problem. Hence, exact cause-and-effect relationships need to be established by incorporating GIS, GPS, and RS-generated data as well as possible interactions. Exploiting the potential of PA technologies in sustainable ways depends on whether or not we first ask ''''Are we doing the right thing?'''' (strategic) as opposed to ''''Are we doing it right?'''' (tactical).     

Molecular Analysis of the Interactions between Cyst Nematodes and Their Hosts

In order to complete its life cycle, a cyst nematode must stimulate the production of a specialized syncytial feeding site within host root tissues. This process is characterized by major changes in local root morphology, including enlargement of affected nuclei and nucleoli, cell wall degradation, and proliferation of subcellular organelles. At the molecular level very little is known about the processes involved in this host response, but recent evidence suggests that cyst nematodes are able to regulate specific host genes. The host-parasite model system provided by Arabidopsis thaliana and Heterodera schachtii will be fundamental to our future understanding of the formation of syncytia. Molecular biology now offers us the opportunity to study this complex host-parasite interaction in great detail. A better understanding of the host genes regulated by cyst nematodes and the mechanisms by which this regulation is achieved will facilitate the engineering of crop cultivars that possess novel forms of resistance to these adept parasites.     

Identification of Cyst Nematodes of Agronomic and Regulatory Concern with PCR-RFLP of ITS1

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nucleotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from the same individual, between individuals, and between isolates. PCR-RFLP detected several restriction site differences between Indian and U.S. isolates. The basis for the restriction site differences between isolates from India and the U.S. appeared to be the result of additional, variant ITS1 regions amplified from the U.S. isolates, which were not found in the three India isolates. PCR-RFLP from individuals of the U.S. isolates created a composite pattern derived from several ITS1 types. A second primer set was specifically designed to permit discrimination between soybean (H. glycines) and sugar beet (H. schachtii) cyst nematodes. Fok I digestion of amplified product from soybean cyst nematode isolates displayed a uniform pattern, readily discernible from the pattern of sugar beet and clover cyst nematode (H. trifolii).     

Improving the Accuracy of Sampling Field Plots for Plant-Parasitic Nematodes

The validity of nematode data from field experiments depends largely on how well samples represent the nematode population. Data from an intensive sampling of three field plots before and after spring cultivation were used to compare eight simulated sampling schemes. Average deviation from the plot mean ranged from 10% to 34% before cultivation and from 7% to 16% after cultivation. Samples taken from only the plant row erred most before cultivation but were comparable to other schemes after cultivation. Several schemes achieved a 25% deviation or less in 90% of the sample simulations. Sampling a nematode population usually involves subsampling a composite bulk sample, however, and this increases error by an estimable amount. A random sample with 35 cores and four random subsamples estimated mean plot densities within 25% with probabilities ranging from 0.77 to 0.85. The probability of a sample-subsample combination coming within a specified percent error of the true mean can be extended cautiously to any field mean and variance more-or-less independent of species and area using formulae presented herein. The most economical method of increasing sample accuracy was to increase the number of soil cores.     

Early Crop Root Destruction for Management of Tobacco Cyst Nematodes

Prompt tillage after crop harvest was investigated as a cultural control for the tobacco cyst nematode, Globodera tabacum tabacum, on stalk-cut broadleaf cigar wrapper tobacco. Stalk stumps and roots remaining after harvest were destroyed by tilling immediately or from 2 to 6 wk after harvest in field experiments over 4 yr. Cyst nematode Pf/Pi ratios ranged from 0.65 to 1.62 when plants were tilled immediately after harvest and 1.13 to 5.88 when tillage was delayed. Nematode population development was monitored by inoculating plants in pots placed in fields with J2 in eggs and sampling over time (8 to 18 wk). Three generations per year were observed, and G. t. tabacum generation time was as short as 6 wk for each generation. Destroying stalks and root systems remaining after harvesting stalk-cut broadleaf cigar wrapper tobacco removes the host to preclude development of nematodes at the end of the second and entire third generation. Early tillage resulted in consistently lower tobacco cyst nematode populations than allowing viable roots to remain in fields for an additional 8 to 18 wk. This management tactic reduces the need for nematicide application to slow nematode population increases over time and can reduce losses due to infection by G. t. tabacum.     

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 μg benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 μg/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 μg/ml, but some hyphae grew on medium amended with 1,000 μg/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 μg/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.     

Entomopathogenic Nematodes Are Not an Alternative to Fenamiphos for Management of Plant-Parasitic Nematodes on Golf Courses in Florida

With the cancellation of fenamiphos in the near future, alternative nematode management tactics for plant-parasitic nematodes (PPN) on golf courses need to be identified. The use of entomopathogenic nematodes (EPN) has been suggested as one possible alternative. This paper presents the results of 10 experiments evaluating the efficacy of EPN at managing PPN on turfgrasses and improving turf performance. These experiments were conducted at various locations throughout Florida over the course of a decade. In different experiments, different EPN species were tested against different species of PPN. Separate experiments evaluated multiple rates and applications of EPN, compared different EPN species, and compared single EPN species against multiple species of PPN. In a few trials, EPN were associated with reductions in certain plant-parasite species, but in other trials were associated with increases. In most trials, EPN had no effect on plant parasites. Because EPN were so inconsistent in their results, we conclude that EPN are not acceptable alternatives to fenamiphos by most turf managers in Florida at this time.     

Hatching Behavior of Potato Cyst Nematodes from the Canary Islands

The present work investigated early hatching differences in naturally occuring field populations and newly reared populations of potato cyst nematodes from the Canary Islands. Hatching behavior of the two species appears to be distinct, with more juveniles hatched from G. pallida that hatch earlier and over a shorter time than G. rostochiensis. The hatching rate of 3-year-old PCN populations was more than double (mean 44.5% ñ 1) that shown by newly reared populations (mean 19.1% ñ 12.5), and those that could be classified as pathotype Pa 1 (Pa 1 and P 13) were found to hatch particularly poorly. Significant differences were also observed in the juveniles released in tap water between newly reared populations of both species, with mean hatch significantly higher for G. rostochiensis. The results are discussed in relation to the implication that these findings may have for competition between the two species of PCN in the field.     

Portable Cyst Extractor: Detecting Cyst Nematodes in the Field

For on-site detection of cysts, a portable cyst extraction kit was constructed from nine readily available items. The portable cyst extractor detected cysts in a range of 1-117 cysts/100 g soil from 42 fields. Samples processed by this kit in fields were clean and easy to examine, possibly because the kit is a compact version of the standard wet-sieving technique used in the laboratory. The portable cyst extractor has several advantages over traditional survey methods: i) diagnosis of cyst infestations in the field accurately and rapidly most of the year and ii) reduction in the labor of samplings and transportation of soil samples.     

Combinations of Biocontrol Agents for Management of Plant-Parasitic Nematodes and Soilborne Plant-Pathogenic Fungi

Numerous microbes are antagonistic to plant-parasitic nematodes and soilborne plant-pathogenic fungi, but few of these organisms are commercially available for management of these pathogens. Inconsistent performance of applied biocontrol agents has proven to be a primary obstacle to the development of successful commercial products. One of the strategies for overcoming inconsistent performance is to combine the disease-suppressive activity of two (or more) beneficial microbes in a biocontrol preparation. Such combinations have potential for more extensive colonization of the rhizosphere, more consistent expression of beneficial traits under a broad range of soil conditions, and antagonism to a larger number of plant pests or pathogens than strains applied individually. Conversely, microbes applied in combination also may have antagonistic interactions with each other. Increased, decreased, and unaltered suppression of the target pathogen or pest has been observed when biocontrol microbes have been applied in combination. Unfortunately, the ecological basis for increased or decreased suppression has not been determined in many cases and needs further consideration. The complexity of interactions involved in the application of multiple organisms for biological control has slowed progress toward development of successful formulations. However, this approach has potential for overcoming some of the efficacy problems that occur with application of individual biocontrol agents.     

Alternatives to Fenamiphos for Management of Plant-Parasitic Nematodes on Bermudagrass

Plant-parasitic nematodes can be very damaging to turfgrasses. The projected cancellation of the registration for fenamiphos in the near future has generated a great deal of interest in identifying acceptable alternative nematode management tactics for use on turfgrasses. Two field experiments were conducted to evaluate the effectiveness of repeated applications of several commercially available nematicides and root biostimulants for reducing population densities of plant-parasitic nematodes and (or) promoting health of bermudagrass in nematode-infested soil. One experimental site was infested with Hoplolaimus galeatus and Trichodorus obtusus, the second with Belonolaimus longicaudatus. In both trials, none of the experimental treatments reduced population densities (P ≤ 0.1) of plant-parasitic nematodes, or consistently promoted turf visual performance or turf root production. Nematologists with responsibility to advise turf managers regarding nematode management should thoroughly investigate the validity of product claims before advising clientele in their use.     

Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode

The isolation of the nematode-resistance gene Gpa2 in potato is described, and it is demonstrated that highly homologous resistance genes of a single resistance-gene cluster can confer resistance to distinct pathogen species. Molecular analysis of the Gpa2 locus resulted in the identification of an R-gene cluster of four highly homologous genes in a region of approximately 115 kb. At least two of these genes are active: one corresponds to the previously isolated Rx1 gene that confers resistance to potato virus X, while the other corresponds to the Gpa2 gene that confers resistance to the potato cyst nematode Globodera pallida. The proteins encoded by the Gpa2 and the Rx1 genes share an overall homology of over 88% (amino-acid identity) and belong to the leucine-zipper, nucleotide-binding site, leucine-rich repeat (LZ-NBS-LRR)-containing class of plant resistance genes. From the sequence conservation between Gpa2 and Rx1 it is clear that there is a direct evolutionary relationship between the two proteins. Sequence diversity is concentrated in the LRR region and in the C-terminus. The putative effector domains are more conserved suggesting that, at least in this case, nematode and virus resistance cascades could share common components. These findings underline the potential of protein breeding for engineering new resistance specificities against plant pathogens in vitro.     

Resolution of natural hatching factors for golden potato cyst nematode, Globodera rostochiensis

The hatching responses of Globodera rostochiensis (golden potato cyst nematode) to purified and partially-purified preparations of natural (including the potato glycoalkaloids solanine and α-chaconine) and artificial hatching factors (HFs) were bimodal. At least 10 HFs, mostly anionic, were resolved from potato root leachate by a combination of gel permeation and ion-exchange chromatography. Whereas potato roots were the principal source of HFs, haulm leachate also contained such chemicals. Root leachate from aseptically-grown potato plants lacked several HFs which were present in conventionally-produced leachate.     

Inbreeding and Hybridizing Cyst Nematodes on Pruned Soybeans in Petri Plates

Inbred nematodes propagated on a selecting host are likely to have homozygous genes of interest for investigating the genetics of host-parasite associations. A technique is presented to inbreed soybean cyst nematodes, by sibling matings at each generation, and to cross inbred lines. Soybean seedlings with severely trimmed cotyledons survive well on 0.8% agar. Eggs from a single female are incubated in water in a microtiter well. Virgin as well as mated females result from inoculation of two juveniles per root. Sibling males from the same source are produced by mass inoculations of eggs. Males are added individually to unmated females. Overall success for fertile females was 14% in 1,368 isolations. Three generations of inbreeding by siblings were achieved using nematodes from two populations that differ in their ability to reproduce on differential soybeans. Hybrids from crosses of the two inbred lines tested on differential hosts showed that the influence of Population 1 (selected and inbred on PI 209332) is greater than that of Population 2 (selected and inbred on PI 89772). Reciprocal crosses suggest that the influence of males is stronger than that of females in determining host specificity of F₁ offspring in these crosses. Our technique is simple and effective for inbreeding and crossing soybean cyst nematodes.