Interrelationships of Rotylenchulus reniformis with Rhizoctonia solani on Cotton

The interrelationships between reniform nematode (Rotylenchulus reniformis) and the cotton (Gossypium hirsutum) seedling blight fungus (Rhizoctonia solani) were studied using three isolates of R. solani, two populations of R. reniformis at multiple inoculum levels, and the cotton cultivars Dehapine 90 (DP 90) and Dehapine 41 (DP 41). Colonization of cotton hypocotyl tissue by R. solani resulted in increases (P ≤ 0.05) in nematode population densities in soil and in eggs recovered from the root systems in both 40- and 90-day-duration experiments. Increases in soil population densities resulted mainly from increases in juveniles. Enhanced reproduction of R. reniformis in the presence of R. solani was consistent across isolates (1, 2, and 3) of R. solani and populations (1 and 2) and inoculum levels (0.5, 2, 4, and 8 individuals/g of soil) of R. reniformis, regardless of cotton cultivar (DP 90 or DP 41). Severity of seedling blight was not influenced by the nematode. Rhizoctonia solani caused reductions (P ≤ 0.05) in cotton growth in 40- and 90-day periods. Rotylenchulus reniformis reduced cotton growth at 90 days. The relationship between nematode inoculum levels and plant growth reductions was linear. At 90 days, the combined effects of these pathogens were antagonistic to plant growth.     

Influence of Nonhost Plants on Population Decline of Rotylenchulus reniformis

The influence of Chloris gayana, Crotalaria juncea, Digitaria decumbens, Tagetes patula, and a chitin-based soil amendment on Hawaiian populations of Rotylenchulus reniformis was examined. Chloris gayana was a nonhost for R. reniformis. The nematode did not penetrate the roots, and in greenhouse and field experiments, C. gayana reduced reniform nematode numbers at least as well as fallow. Tagetes patula was a poor host for reniform nematode and reduced reniform nematode numbers in soil better than did fallow. Crotalaria juncea was a poor host for R. reniformis, and only a small fraction of the nematode population penetrated the roots. Crotalaria juncea and D. decumbens reduced reniform nematode populations at least as well as fallow. A chitin-based soil amendment, applied at 2.24 t/ha to fallow soil, did not affect the population decline of reniform nematode.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Fluopyram

Fluopyram is a succinate dehydrogenase inhibitor (SDHI) fungicide that is being evaluated as a seed treatment and in-furrow spray at planting on row crops for management of fungal diseases and its effect on plant-parasitic nematodes. Currently, there are no data on nematode toxicity, nematode recovery, or effects on nematode infection for Meloidogyne incognita or Rotylenchulus reniformis after exposure to low concentrations of fluopyram. Nematode toxicity and recovery experiments were conducted in aqueous solutions of fluopyram, while root infection assays were conducted on tomato. Nematode paralysis was observed after 2 hr of exposure at 1.0 µg/ml fluopyram for both nematode species. Using an assay of nematode motility, 2-hr EC₅₀ values of 5.18 and 12.99 µg/ml fluopyram were calculated for M. incognita and R. reniformis, respectively. Nematode recovery in motility was greater than 50% for M. incognita and R. reniformis 24 hr after nematodes were rinsed and removed from a 1-hr treatment of 5.18 and 12.99 µg/ml fluopyram, respectively. Nematode infection of tomato roots was reduced and inversely proportional to 1-hr treatments with water solutions of fluopyram at low concentrations, which ranged from 1.3 to 5.2 µg/ml for M. incognita and 3.3 to 13.0 µg/ml for R. reniformis. Though fluopyram is nematistatic, low concentrations of the fungicide were effective at reducing the ability of both nematode species to infect tomato roots.     

Effect of Controlled Cold Storage on Recovery of Rotylenchulus reniformis from Naturally Infested Soil

Rotylenchulus reniformis is rapidly becoming the most economically important pest associated with cotton in the southeastern United States. Incentive programs have been implemented to support sampling of production fields to determine the presence and abundance of R. reniformis. These sampling programs have dramatically increased the number of soils samples submitted to nematology laboratories during autumn. The large numbers of samples overwhelm most labs and require placement in cold storage until extraction. Therefore, the objective of this study was to examine the length of time soils infested with R. reniformis can be stored before nematode extraction without compromising the accuracy of estimates of population densities. A sandy loam and a silty loam were the two cotton production soils used in this study. Rotylenchulus reniformis numbers decreased 61%during the first 180 days of storage in both soils. Rotylenchulus reniformis numbers from the initial sampling through 180 days decreased as a linear function. The decline of R. reniformis numbers during storage was estimated as 0.28% of the population lost daily from the maximum population through 180 days. The diminution of nematode numbers from 180 through 1,080 days in storage continued, but at a slower rate. Numbers of R. reniformis declined to less than 89%, 93%, and 99% of the initial population within 360, 720, and 1,080 days, respectively, of storage. The reduction of R. reniformis numbers over 180 days can be adjusted, allowing a more accurate estimation of R. reniformis levels in soil samples stored at 4 °C.     

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin

Avermectins are macrocyclic lactones produced by Streptomyces avermitilis. Abamectin is a blend of B_1a and B_1b avermectins that is being used as a seed treatment to control plant-parasitic nematodes on cotton and some vegetable crops. No LD₅₀ values, data on nematode recovery following brief exposure, or effects of sublethal concentrations on infectivity of the plant-parasitic nematodes Meloidogyne incognita or Rotylenchulus reniformis are available. Using an assay of nematode mobility, LD₅₀ values of 1.56 μg/ml and 32.9 μg/ml were calculated based on 2 hr exposure for M. incognita and R. reniformis, respectively. There was no recovery of either nematode after exposure for 1 hr. Mortality of M. incognita continued to increase following a 1 hr exposure, whereas R. reniformis mortality remained unchanged at 24 hr after the nematodes were removed from the abamectin solution. Sublethal concentrations of 1.56 to 0.39 μg/ml for M. incognita and 32.9 to 8.2 μg/ml for R. reniformis reduced infectivity of each nematode on tomato roots. The toxicity of abamectin to these nematodes was comparable to that of aldicarb.     

Competition of Meloidogyne incognita and Rotylenchulus reniformis on Cotton Following Separate and Concomitant Inoculations

It has been hypothesized Rotylenchulus reniformis (Rr) has a competitive advantage over Meloidogyne incognita (Mi) in the southeastern cotton production region of the United States. This study examines the reproduction and development of Meloidogyne incognita (Mi) and Rotylenchulus reniformis (Rr) in separate and concomitant infections on cotton. Under greenhouse conditions, cotton seedlings were inoculated simultaneously with juveniles (J2) of M. incognita and vermiform adults of R. reniformis in the following ratios (Mi:Rr): 0:0, 100:0, 75:25, 50:50, 25:75, and 0:100. Soil populations of M. incognita and R. reniformis were recorded at 3, 6, 9, 14, 19, 25, 35, 45, and 60 days after inoculations. At each date, samples were taken to determine the life stage of development, number of egg masses, eggs per egg mass, galls, and giant cells or syncytia produced by the nematodes. Meloidogyne incognita and R. reniformis were capable of initially inhibiting each other when the inoculum ratio of one species was higher than the other. In concomitant infections, M. incognita was susceptible to the antagonistic effect of R. reniformis. Rotylenchulus reniformis affected hatching of M. incognita eggs, delayed secondary infection of M. incognita J2, reduced the number of egg masses produced by M. incognita, and reduced J2 of M. incognita 60 days after inoculations. In contrast, M. incognita reduced R. reniformis soil populations only when its proportion in the inoculum ratio was higher than that of R. reniformis. Meloidogyne incognita reduced egg masses produced by R. reniformis, but not production of eggs and secondary infection.     

Sterile Culture of Rotylenchulus reniformis on Tomato Root with Gellan Gum as a Supporting Medium

Rotylenchulus reniformis was repeatedly propagated in sterile excised tomato roots growing on modified White''s medium with gellan gum as the support. Gellan gum provided an optically clear support medium that could be liquified by adding 5 mM disodium ethylenediaminetetraacetate (EDTA) to facilitate nematode extraction. Liquefaction of the gellan-gum medium by EDTA allowed efficient recovery of eggs and vermiform stages of R. reniformis. Extraction efficiency was quantified with Radopholus similis as a test organism. The efficiency of extracting R. similis from the gellan gum did not vary with the concentrations of EDTA tested.     

Suppression of Rotylenchulus reniformis on Cotton by the Nematophagous Fungus ARF

The reniform nematode, Rotylenchulus reniformis Linford &Oliveira, has become a serious threat to cotton (Gossypium hirsutum L.) production in the United States during the past decade. The objective of this study is to isolate fungi from eggs of R. reniformis and select potential biological control agents for R. reniformis on cotton. Soil samples were collected from cotton fields located in Jefferson County, Arkansas. Eight genera of fungi were included in the 128 fungal isolates obtained, and among them were five strains of the nematophagous fungus ARF. The mtDNA RFLP pattern, colony growth characteristics, and pathogenicity indicate the five ARF isolates represent one described strain and one new strain. Light and electron microscopic observations suggest ARF is an active parasite of R. reniformis, with parasitism ranging from 48% to 79% in in vitro tests. Three greenhouse experiments demonstrated ARF successfully suppressed the number of reniform nematodes during the first and second generation of the nematode. Reductions in numbers of R. reniformis on the roots for the seven application rates of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ARF were 87%, 92%, 94%, 96%, 97%, 98%, and and 98%, respectively.     

Effect of Temperature on Infection and Survival of Rotylenchulus reniformis

From infestation of lettuce with preinfective females to egg deposition, populations of Rotylenchulus reniformis from Baton Rouge, Louisiana; Lubbock and Weslaco, Texas; and Mayaguez, Puerto Rico, required 41, 13, 7, and 7 days at 15, 20, 25, and 34 C, respectively. No nematode infection occurred at 10 C with any R. reniformis population, and the population from Puerto Rico did not reproduce at 15 C. Nematode survival was not influenced by temperature, since populations from Texas and Louisiana survived for 6 months without a host at - 5 , - 1 , 4, and 25 C. Survival of R. reniformis was substantially influenced by soil moisture. Soil moistures greater than 7% (< 1 bar) aided nematode survival at storage temperature of 25 C, whereas moisture adversely affected nematode survival below freezing. Soil moisture below 4% (> 15 bars) favored nematode survival below freezing but adversely affected nematodes in soils stored at 25 C. Soil moisture effects on nematode survival were less accentuated at 4 and 0 C.     

The Effect of Soil Texture and Irrigation on Rotylenchulus reniformis and Cotton

The reniform nematode, Rotylenchulus reniformis, is the most damaging nematode pathogen of cotton in Alabama. Soil texture is currently being explored as a basis for the development of economic thresholds and management zones within a field. Trials to determine the reproductive potential of R. reniformis as influenced by soil type were conducted in microplot and greenhouse settings during 2008 to 2010. Population density of R. reniformis was significantly influenced by soil texture and exhibited a general decrease with increasing median soil particle size (MSPS). As the MSPS of a soil increased from 0.04 mm in clay soil to > 0.30 mm in very fine sandy loam and sandy loam soils, R. reniformis numbers decreased. The R. reniformis population densities on all soil types were also greater with irrigation. Early season cotton development was significantly affected by increasing R. reniformis Pi, with plant shoot-weight-to-root-weight ratios increasing at low R. reniformis Pi and declining with increasing R. reniformis Pi. Plant height was increased by irrigation throughout the growing season. The results suggests that R. reniformis will reach higher population densities in soils with smaller MSPS; however, the reduction in yield or plant growth very well may be no greater than in a soil that is less preferential to the nematode.     

Using FAME Analysis to Compare, Differentiate, and Identify Multiple Nematode Species

We have adapted the Sherlock^® Microbial Identification system for identification of plant parasitic nematodes based on their fatty acid profiles. Fatty acid profiles of 12 separate plant parasitic nematode species have been determined using this system. Additionally, separate profiles have been developed for Rotylenchulus reniformis and Meloidogyne incognita based on their host plant, four species and three races within the Meloidogyne genus, and three life stages of Heterodera glycines. Statistically, 85% of these profiles can be delimited from one another; the specific comparisons between the cyst and vermiform stages of H. glycines, M. hapla and M. arenaria, and M. arenaria and M. javanica cannot be segregated using canonical analysis. By incorporating each of these fatty acid profiles into the Sherlock^® Analysis Software, 20 library entries were created. While there was some similarity among profiles, all entries correctly identified the proper organism to genus, species, race, life stage, and host at greater than 86% accuracy. The remaining 14% were correctly identified to genus, although species and race may not be correct due to the underlying variables of host or life stage. These results are promising and indicate that this library could be used for diagnostics labs to increase response time.     

Vertical Distribution of Rotylenchulus reniformis in Cotton Fields

The possible impact of Rotylenchulus reniformis below plow depth was evaluated by measuring the vertical distribution of R. reniformis and soil texture in 20 symptomatic fields on 17 farms across six states. The mean nematode population density per field, 0 to 122 cm deep, ranged from 0.4 to 63 nematodes/g soil, and in 15 fields more than half of the R. reniformis present were below 30.5 cm, which is the greatest depth usually plowed by farmers or sampled by consultants. In 11 fields measured, root density was greatest in the top 15 cm of soil; however, roots consistently penetrated 92 to 122 cm deep by midseason, and in five fields in Texas and Louisiana the ratio of nematodes to root-length density within soil increased with depth. Repeated sampling during the year in Texas indicated that up to 20% of the nematodes in soil below 60 cm in the fall survived the winter. Differences between Baermann funnel and sugar flotation extraction methods were not important when compared with field-to-field differences in nematode populations and field-specific vertical distribution patterns. The results support the interpretation that R. reniformis below plow depth can significantly impact diagnosis and treatment of cotton fields infested with R. reniformis.     

Suppression of Rotylenchulus reniformis 122-cm Deep Endorses Resistance Introgression in Gossypium

Nine sources of resistance to Rotylenchulus reniformis in Gossypium (cotton) were tested by measuring population density (Pf) and root-length density 0 to 122 cm deep. A Pf in the plow layer less than the autumn sample treatment threshold used by consultants was considered the minimum criterion for acceptable resistance, regardless of population density at planting (Pi). Other criteria were ample roots and a Pf lower than on the susceptible control, as in pot studies. In a Texas field in 2001 and 2002, no resistant accessions had Pf less than the control but all did in microplots into which nematodes from Louisiana were introduced. An environmental chamber experiment ruled out nematode genetic variance and implicated unknown soil factors. Pf in field experiments in Louisiana, Mississippi, and Alabama were below threshold for zero, six and four of the accessions and above threshold in the control. Gossypium arboreum A2–87 and G. barbadense GB-713 were the most resistant accessions. Results indicate that cultivars developed from these sources will suppress R. reniformis populations but less than in pots in a single season.     

Intercropping Cover Crops with Pineapple for the Management of Rotylenchulus reniformis

Effect of cover crops intercropped with pineapple (Ananas comosus) on Rotylenchulus reniformis population densities and activity of nematode-trapping fungi (NTF) were evaluated in two cycles of cover crop and pineapple. Sunn hemp (Crotalaria juncea), rapeseed (Brassica napus), African marigold (Tagetes erecta), or weeds were intercropped with pineapples. Beds planted with sunn hemp or rapeseed had lower population densities of R. reniformis than African marigold, weeds, or pineapple plots during cover crop growth, and the subsequent pineapple-growing periods. Rapeseed was a good host to Meloidogyne javanica and resulted in high population densities of M. javanica in the subsequent pineapple crop. Fireweed (Erigeron canadensis) occurred commonly and was a good host to R. reniformis. Bacterivorous nematode population densities increased (P ≤ 0.05) most in sunn hemp, especially early after planting. Nematode-trapping fungi required a long period to develop measurable population densities. Population densities of NTF were higher in cover crops than weeds or pineapples during the first crop cycle (P < 0.05). Although pineapple produced heavier fruits following sunn hemp than in the other treatments (P < 0.05), commercial yields were not different among rapeseed, weed, and sunn hemp treatments.     

Interaction of Rotylenchulus reniformis with Seedling Disease Pathogens of Cotton

The impact of 10 Fusarium species in concomitant association with Rotylenchulus reniformis on cotton seedling disease was examined under greenhouse conditions. In experiment 1, fungal treatments consisted of Fusarium chlamydosporum, F. equiseti, F. lateritium, F. moniliforme, F. oxysporum, F. oxysporum f.sp. vasinfectum, F. proliferatum, F. semitectum, F. solani, and F. sporotrichioides; Rhizoctonia solani; and Thielaviopsis basicola. The experimental design was a 2 × 14 factorial consisting of the presence or absence of R. reniformis and the 12 fungal treatments plus two controls in autoclaved field soil. In experiment 2, the same fungal and nematode treatments were examined in autoclaved or non-autoclaved soil. This experimental design was a 2 × 2 × 14 factorial consisting of field or autoclaved soil, presence or absence of R. reniformis, and the 12 fungal treatments plus two controls. In both tests, Fusarium oxysporum f. sp. vasinfectum, F. solani, R. solani, and T. basicola consistently displayed extensive root and hypocotyl necrosis that was more severe (P ≤ 0.05) in the presence of R. reniformis. Soil treatment (autoclaved vs. non-autoclaved) influenced the impact of the Fusarium species on cotton seedling disease, with disease being more severe in the autoclaved soil. Rotylenchulus reniformis reproduction on cotton seedlings was greater in field soil compared to autoclaved soil (P ≤ 0.05). This study suggests the importance of Fusarium species and R. reniformis in cotton seedling disease.     

Stage-specific Monoclonal Antibodies to the Potato Cyst Nematode Globodera pallida Stone (Behrens)

Using standard hybridoma technology and hierarchical screening, monoclonal antibodies (MAbs) were obtained with specific reactivity against two developmental stages of Globodera pallida. The procedure was based on enzyme-linked immunosorbent assay (ELISA) with homogenates prepared from second-stage juveniles, young adult females, and potato roots. Hybridomas were formed by fusing myelomas with splenocytes derived from mice immunized with either infective juveniles or females of G. pallida. About 600 hybridoma lines were screened from the fusion involving the mouse immunized with juveniles. Two MAbs (LJMAbl &2) were identified with high reactivity toward second-stage juveniles but no reactivity with either potato roots or females of G. pallida. A total of 630 cell lines was screened from the corresponding fusion involving the spleen of a mouse receiving immunogens from adult female nematodes. One MAb (LFMAbl) was obtained with the required specificity against only adult female G. pallida. This work extends the application of monoclonal antibodies in nematology from valuable probes for research and species identification to recognition of developmental stages. These specific MAbs have potential value in plant breeding programs for screening for resistant lines unable to support nematode development.     

Influence of Rhizoctonia solani on Egg Hatching and Infectivity of Rotylenchulus reniformis

The effects of culture filtrates of Rhizoctonia solani and root exudates of R. solani-infected cotton (Gossypium hirsutum) seedlings on hatching of eggs and infectivity of females of Rotylenchulus reniformis were evaluated in an attempt to account for the enhanced nematode reproduction observed in the presence of this fungus. Crude filtrates of R. solani cultures growing over sterile, deionized distilled water did not affect egg hatching. Exudates from roots of cotton seedlings increased hatching of R. reniformis eggs over that observed in water controls. Exudates from cotton seedling roots not infected or infected with R. solani did not differ in their effect on egg hatching. However, infection of cotton seedlings by reniform females was increased in the presence of R. solani, resulting in the augmented egg production and juvenile population densities in soil observed in greenhouse studies.     

Impact of Soil Texture on the Reproductive and Damage Potentials of Rotylenchulus reniformis and Meloidogyne incognita on Cotton

The effects of soil type and initial inoculum density (Pi) on the reproductive and damage potentials of Meloidogyne incognita and Rotylenchulus reniformis on cotton were evaluated in microplot experiments from 1991 to 1993. The equilibrium nematode population density for R. reniformis on cotton was much greater than that of M. incognita, indicating that cotton is a better host for R. reniformis than M. incognita. Reproduction of M. incognita was greater in coarse-textured soils than in fine-textured soils, whereas R. reniformis reproduction was greatest in a Portsmouth loamy sand with intermediate percentages of clay plus silt. Population densities of M. incognita were inversely related to the percentage of silt and clay, but R. reniformis was favored by moderate levels of clay plus silt (ca. 28%). Both M. incognita races 3 and 4 and R. reniformis effected suppression of seed-cotton yield in all soil types evaluated. Cotton-yield suppression was greatest in response to R. reniformis at high Pi. Cotton maturity, measured as percentage of open bolls at different dates, was affected by the presence of nematodes in all 3 years.