Stem Nematode-Fusarium Wilt Complex in Alfalfa as Related to Irrigation Management at Harvest Time

A high moisture level in the top 10 cm of soil at time of cutting of alfalfa increased the incidence of plant mortality and Fusarium wilt in soil infested with Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis in greenhouse and field microplot studies. Ranger alfalfa, susceptible to both D. dipsaci and F. oxysporum f. sp. medicaginis, was less persistent than Moapa 69 (nematode susceptible and Fusarium wilt resistant) and Lahontan alfalfa (nematode resistant with low Fusarium wilt resistance). In the greenhouse, the persistence of Ranger, Moapa 69, and Lahontan alfalfa plants was 46%, 64%, and 67% respectively, in nematode + fungus infested soil at high soil moisture at time of cutting. This compared to 74%, 84%, and 73% persistence of Ranger, Moapa 69, and Lahontan, respectively, at low soil moisture at time of cutting. Shoot weights as a percentage of uninoculated controls at the high soil moisture level were 38%, 40%, and 71% for Ranger, Moapa 69, and Lahontan, respectively. Low soil moisture at time of cutting negated the effect D. dipsaci on plant persistence and growth of subsequent cuttings, and reduced Fusarium wilt of plants in the nematode-fungus treatment; shoot weights were 75%, 90%, and 74% of uninoculated controls for Ranger, Moapa 69, and Lahontan. Similar results were obtained in the field microplot study, and stand persistence and shoot weights were less in nematode + fungus-infested soil at the high soil-moisture level (early irrigation) than at the low soil-moisture level (late irrigation).     

Pathological Relationship of Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis on alfalfa

Ditylenchus dipsaci and Fusarium oxysporum f. sp. medicaginis synergistically affected the mortality and plant growth of Ranger alfalfa, a cultivar susceptible to stem nematode and Fusarium wilt. The nematode-fungus relationship had an additive effect on mortality and plant growth of Lahontan (nematode resistant and Fusarium wilt susceptible) and of Moapa 69 (nematode susceptible and Fusarium wilt resistant). Mortality rates were 13, 16, 46, and 49% for Ranger; 4, 18, 26, and 28% for Lahontan; and 19, 10, 32, and 30% for Moapa 69 inoculated with D. dipsaci, F. oxysporum f. sp. medicaginis, and simultaneously and sequentially with D. dipsaci and F. oxysporum f. sp. medicaginis, respectively. Shoot weights as a percentage of uninoculated controls for the same treatments were 52, 84, 26, and 28%, for Ranger; 74, 86, 64, and 64% for Lahontan; and 50, 95, 44, and 39% for Moapa 69. Plant growth suppression was related to vascular bundle infection and discoloration of alfalfa root tissue. Disease severity and plant growth of alfalfa did not differ with simultaneous or sequential inoculations of the two pathogens. Fusarium oxysporum f. sp. medicaginis affected alfalfa growth but not nematode reproduction.     

Effects of Environmental Factors and Cultural Practices on Parasitism of Alfalfa by Ditylenchus dipsaci

Cool humid weather enhanced development and reproduction of Ditylenchus dipsaci in alfalfa in laboratory and field studies in Utah. Relative humidity and nematode reproduction were positively correlated (P < 0.05), whereas air temperature and nematode reproduction were negatively correlated (P < 0.05). The greatest number of nematodes per gram of alfalfa tissue was found in nondormant Moapa alfalfa tissue at St. George during April, whereas the greatest numbers of nematodes were found in dormant Ranger alfalfa in June at West Jordan and Smithfield. There was 100% invasion of both resistant Lahontan and susceptible Ranger alfalfa plants at soil moisture levels of 61-94% field capacity. Fall burning of alfalfa to control weeds reduced, and spring burning increased, the incidence of invaded plants, nematodes per gram of plant tissue, and the mortality of susceptible Ranger (P < 0.01) and Moapa (P < 0.01) alfalfa plants over that of plants in nonburned control plots. Fall burning also reduced and spring burning increased the incidence of invaded plants (P < 0.05), but had no influence on nematodes per gram of plant tissue or the mortality of resistant Lahontan and Nevada Synthetic XX alfalfa over those of plants in control plots.     

A Nematicide Seed Treatment to Control Ditylenchus dipsaci on Seedling Alfalfa

Three nematicides were evaluated as seed treatments to control the alfalfa stem nematode (Ditylenchus dipsaci) on seedling alfalfa. Alfalfa seeds were soaked for 10 hours in a 0.5% (formulated by weight) concentration of either carbofuran, phenamiphos or oxamyl in acetone with no adverse effect on seed germination. All three treatments decreased nematode damage and increased survival of ''Ranger'' (susceptible) and ''Lahontan'' (resistant) alfalfa plants, when seeds were planted in soil infested with D. dipsaci. Mean live plant counts after 6 weeks in the untreated control, acetone alone, carbofuran, phenamiphos, and oxamyl treatments, respectively, were 4.3, 6.3, 19.0, 19.8, and 19.0 for Lahontan and 4.5, 1.5, 18.5, 19.3, and 18.0 for Ranger from 20 seeds/pot. Nematicide seed treatments resulted in significantly healthier Ranger alfalfa plants 4 months after planting. The combination of seed treatment and host resistance may provide a means of establishing alfalfa in an alfalfa monocropped system where soil populations of D. dipsaci are high.     

Seasonal Fluctuations of Soil and Tissue Populations of Ditylenchus dipsaci and Aphelenchoides ritzemabosi in Alfalfa

Population dynamics of A. ritzemabosi and D. dipsaci were studied in two alfalfa fields in Wyoming. Symptomatic stem-bud tissue and root-zone soil from alfalfa plants exhibiting symptoms of D. dipsaci infection were collected at intervals of 3 to 4 weeks. Both nematodes were extracted from stem tissue with the Baermann funnel method and from soil with the sieving and Baermann funnel method. Soil moisture and soil temperature at 5 cm accounted for 64.8% and 61.0%, respectively, of the variability in numbers of both nematodes in soil at the Big Horn field. Also at the Big Horn field, A. ritzemabosi was found in soil on only three of the 14 collection dates, whereas D. dipsaci was found in soil on 12 dates. Aphelenchoides ritzemabosi was found in stem tissue samples on 9 of the 14 sampling dates whereas D. dipsaci was found on all dates. Populations of both nematodes in stem tissue peaked in October, and soil populations of both peaked in January, when soil moisture was greatest. Numbers of D. dipsaci in stem tissue were related to mean air temperature 3 weeks prior to tissue collection, while none of the climatic factors measured were associated with numbers of A. ritzemabosi. At the Dayton field, soil moisture plus soil temperature at 5 cm accounted for 98.2% and 91.4% of the variability in the soil populations of A. ritzemabosi and D. dipsaci, respectively. Aphelenchoides ritzemabosi was extracted from soil at two of the five collection dates, compared to extraction of D. dipsaci at three dates. Aphelenchoides ritzemabosi was collected from stem tissue at six of the seven sampling dates while D. dipsaci was found at all sampling dates. The only environmental factor that was associated with an increase in the numbers of both nematodes in alfalfa stem tissue was total precipitation 1 week prior to sampling, and this occurred only at the Dayton field. Numbers of A. ritzemabosi in stem tissue appeared to be not affected by any of the environmental factors studied, while numbers of D. dipsaci in stem tissue were associated with cumulative monthly precipitation, snow cover at time of sampling, and the mean weekly temperature 3 weeks prior to sampling. Harvesting alfalfa reduced the numbers of A. ritzemabosi at the Big Horn field and both nematodes at the Dayton field.     

Interaction of Ditylenchus dipsaci and Meloidogyne hapla on Resistant and Susceptible Plant Species

Numbers ofDitylenchus dipsaci or Meloidogyne hapla invading Ranger alfalfa, Tender crop bean, Stone Improved tomato, AH-14 sugarbeet, Yellow sweet clover, and Wasatch wheat from single inoculations were not significantly different from numbers by invasion of combined inoculations. D. dipsaci was recovered only from shoot and M. hapla only from root tissue. Combined inoculations did not affect reproduction of either D. dipsaci or M. hapla. D. dipsaci suppressed shoot growth of all species at 15-30 C, and M. hapla suppressed shoot growth of tomato, sugarbeet, and sweet clover at 20, 25, and 30 C. There was a positive correlation (P < 0.05) between shoot and root growth suppression by D. dipsaci on all cultivars except wheat at 20 C and tomato at 30 C. M. hapla suppressed (P < 0.05) root growth of sugarbeet at 20-50 C and wheat at 30 C. Growth suppression was synergistic in combined inoculations of sweet clover shoot growth at 15 C and root growth at 20-30 C, wheat root growth at 15 and 20 C, and tomato root growth at 15-30 C (P < 0.05) D. dipsaci invasions caused mortality of alfalfa and sweet clover at 15-30 C and sugarbeet at 20-30 C. Mortality rates of alfalfa and sweet clover increased synergistically (P < 0.05) from combined inoculations.     

Early Root Response to Meloidogyne incognita in Resistant and Susceptible Alfalfa Cultivars

The early events of Meloidogyne incognita behavior and associated host responses following root penetration were studied in resistant (cv. Moapa 69) and susceptible (cv. Lahontan) alfalfa. Ten-day-old seedlings of alfalfa cultivars were inoculated with second-stage juveniles (J2) and harvested 12, 24, 48, and 72 hours and 7, 14, and 21 days later. Both cultivars supported similar root penetration and initial J2 migration. By 72 hours after inoculation the majority of J2 were amassed inside the vascular cylinder in roots of susceptible Lahontan, while J2 had not entered the vascular cylinder of resistant Moapa 69 and remained clumped at the root apex. Nematode development progressed normally in Lahontan, but J2 were not observed in Moapa 69 after day 7. The greatest differences between RNA translation products isolated from inoculated and uninoculated roots of Lahanton occurred 72 hours after inoculation. Only minor differences in gene expression were observed between inoculated and uninoculated Moapa 69 roots at 72 hours. Comparison of translation products from inoculated versus mechanically wounded Lahontan roots revealed products that were specific to or enhanced in nematode-infected plants. Moapa 69 appears to possess a type of resistance to M. incognita that does not depend on a conventional hypersensitive response.     

Interrelationship of Meloidogyne hapla and Ditylenchus dipsaci on Resistant and Susceptible Alfalfa

Simultaneous inoculations of alfalfa with Meloidogyne hapla larvae and Ditylenchus dipsaci at 16, 20, 24, and 28 C did not depress penetration of either nematode in ''Nev Syn XX'' -a selection resistant to M. hapla and D. dipsaci, ''Vernal 298'' -a selection resistant to M. hapla and susceptible to D. dipsaci, ''Lahontan'' -a cultivar resistant to D. dipsaci and susceptible to M. hapla, and ''Ranger'' -a cultivar susceptible to both M. hapla and D, dipsaci. Infection with D. dipsaci depressed growth of susceptible ''Vernal 298'' and ''Ranger'' at all soil temperatures, except for ''Vernal 298'' at 16 C. Infection with M. hapla alone did not depress growth of any of the alfalfas. A combination of M. hapla and D. dipsaci resulted in a synergistic weight depression on ''Ranger'' at all soil temperatures. Inoculation of the four alfalfas with D. dipsaci 2, 4, 6, and 8 wk before inoculation with M. hapla at 16, 20, 24, and 28 C did not influence the resistance or susceptibility of ''Nev Syn XX,'' ''Lahontan,'' or ''Ranger.'' However, galling of ''Vernal 298'' by M. hapla was affected by soil temperature, plant age, and inoculation with D. dipsaci.     

Differential Reaction of Alfalfa Cultivars to Meloidogyne hapla and M. chitwoodi Populations

Meloidogyne hapla reproduced and suppressed growth (P < 0.05) of susceptible Lahontan and Moapa alfalfa at 15, 20, and 25 C. At 30 C, resistant Nevada Syn XX lost resistance to M. hapla. M. hapla invaded and reproduced on Rhizobium meliloti nodules of Lahontan and Moapa, inducing giant cell formation and structural disorder of vascular bundles of nodules without disrupting bacteroids. At 15, 20, and 25 C a M. chitwoodi population from Utah reproduced on Lahontan, Moapa, and Nevada Syn XX alfalfa, suppressing growth (P < 0.05). Final densities of the Utah M. chitwoodi population were greater (P < 0.05) than those of Idaho and Washington State populations on Lahontan at 15 and 25 C and on Nevada Syn XX at 15 C, but were less consistent and smaller (P < 0.05) than those of M. hapla on Lahontan and Moapa at 20 and 25 C. Inconsistent reproduction of the Utah M. chitwoodi population on alfalfa suggests the possible existence of nematode strains revealed by variability in alfalfa resistance. No reproduction or inconsistent final nematode population densities with no damage were observed on Lahontan, Moapa, and Nevada Syn XX plants grown in soil infested with Idaho and Washington State M. chitwoodi populations.     

Effect of Ditylenchus dipsaci on Alfalfa Mortality,Winterkill, and Yield

Ditylenchus dipsaci-infected and noninfected alfalfa plants in a naturally infested field were studied from July 1980 to September 1982. Forty-one percent of the plants died during the study. Ninety-seven percent of the plants that died were infected with D. dipsaci. Sixty-nine percent of the observed mortality occurred during winter. Forage yield of infected plants was significantly lower than yield of noninfected plants at each harvest. Stored carbohydrates in infected plants were significantly lower than in noninfected plants. In a controlled environment test, significantly greater mortality occurred in frozen severely infected plants than in frozen noninfected plants, while no mortality occurred in severely infected or noninfected plants that were not frozen. Both forage yield and stored carbohydrates were significantly lower in severely infected than noninfected, non-frozen plants. Mortality in greenhouse-grown plants that were transplanted to field plots was significantly greater in D. dipsaci-infected plants than in noninfected plants after one winter.     

Biological Control of Meloidogyne hapla on Alfalfa and Tomato with the Fungus Meria coniospora

This study was to determine whether Arthrobotrys flagrans, A. oligospora, and Meria coniospora would control the root-knot nematode Meloidogyne hapla on alfalfa and tomato. Alfalfa seeds were coated with a fungus-rye powder in 2% cellulose and were planted in infested soil. Three-week-old seedlings from seed treated with M. coniospora had 60% and 58% fewer galls in two experiments than did seedlings from untreated seeds. Numbers of J2 in the soil were not reduced. Plant growth did not improve. When seed of tomato were coated with M. coniospora and planted in M. hapla-infested soil, roots had 34% fewer galls and 47% fewer J2 in the soil at 28 days. After 56 days there was no reduction in J2 numbers. Plant growth did not improve. When roots of tomato transplants were dusted with M. coniospora fungus-rye powder or sprayed with a spore suspension before planting in M. hapla-infested soil, 42% and 35%, respectively, fewer galls developed in 28 days on treated roots than on roots not treated with fungus. The numbers of J2 extracted from roots or recovered from soil were not reduced, however, and plant growth did not improve.     

Efficacy of Oxamyl Coated on Alfalfa Seed with a Polymer Sticker in Pratylenchus and Meloidogyne Infested Soils

A polymer sticker was used as a coating in which oxamyl was applied to seeds of alfalfa cultivar Saranac for the control of Pratylenchus penetrans and Meloidogyne hapla. The sticker, diluted 1:1 (sticker:water) to 1:5, delayed seedling emergence during the first 4 days after planting. By day 13, however, emergence from all sticker treatments was comparable to the control. Shoot growth of seedlings at day 21 was less than that of the control only from seeds coated with a 1:1 dilution; root growth and nodulation were not affected. Sticker-coated seeds absorbed 30-58% as much water in 3.5 hours as was absorbed by uncoated seeds. Oxamyl concentrations of 40-160 mg/ml in a 1:5 sticker : water mixture had no adverse affect on seedling emergence, growth, and nodulation over 3 weeks. Oxamyl at 160 mg/ml was more effective against P. penetrans than M. hapla. Growth of alfalfa in P. penetrans-infested soil was greater than that of the control in each sampling for 11 weeks. The reduction of number of P. penetrans in soil and roots moderated slowly over 11 weeks from 90% to 60%. Shoot and root growth of alfalfa from oxamyl-coated seed in M. hapla-infested soil were greater than those of the control for 7 and 11 weeks, respectively. The reduction in the number of M. hapla in the soil and roots changed from 80% at 7 weeks to 15% at 11 weeks.     

Effect of Hirsutella rhossiliensis on Infection of Potato by Pratylenchus penetrans

We evaluated the ability of the nematode-pathogenic fungus Hirsutella rhossiliensis (Deuteromycotina: Hyphomycetes) to reduce root penetration and population increase of Pratylenchus penetrans on potato. Experiments were conducted at 24 C in a growth chamber. When nematodes were placed on the soil surface 8 cm from a 14-day-old potato cutting, the fungus decreased the number entering roots by 25%. To determine the effect of the fungus on population increase after the nematodes entered roots, we transplanted potato cuttings infected with P. penetrans into Hirsutella-infested and uninfested soil. After 60 days, the total number of nematodes (roots and soil) was 20 ± 4% lower in Hirsutella-infested than in uninfested soil.     

Biology and Pathogenicity of Pratylenchus neglectus on Alfalfa

Pratylenchus neglectus reduced the growth of alfalfa cultivars in greenhouse and growth chamber studies. Inocula (1,000, 5,000 and 10,000 nematodes per plant) reduced shoot dry weights of Ranger by 16, 27, and 40%, of Lahontan by 16, 32, and 40%, and of Nevada Synthetic XX (Nev Syn XX) by 18, 26, and 37%, respectively, at 26 ñ 2 C. Pratylenchus neglectus at 1,000 nematodes per plant reduced Ranger shoot dry weights by 5, 12, 18, and 27%, at 15, 20, 25, and 30 C, respectively, whereas 5,000 nematodes per plant reduced shoot dry weights by 12, 17, 26, and 38%, respectively, at similar temperatures. Reductions in dry root weights were directly related to reductions in shoot growth. At 1,000 nematodes per plant, Ranger root dry weights were reduced by 3, 14, 40, and 40%, whereas 5,000 nematodes per plant reduced root dry weight by 25, 31, 59, and 63%, respectively, at similar temperatures. Similar results were observed on Lahontan and Nev Syn XX at the same inoculum levels and soil temperatures. Nematode reproductive indices (final nematode population per plant divided by initial nematode inoculum per plant) were higher at 1,000 nematodes per plant than at 5,000 nematodes per plant, were positively correlated with temperature, and were unaffected by cultivar.     

Mass Culturing of Ditylenchus dipsaci to Yield Large Quantities of Inoculum

Methods are described for rearing large quantities of Ditylenchus dipsaci on alfalfa tissues. Nematodes and alfalfa seed were disinfected and nematodes were reared in quantities sufficient to provide a continuous supply of inoculum for our alfalfa-breeding program. Nematodes reproduced best in darkness at 20-25 C. Cultures reached maximum numbers in 3-6 wk.     

Differential Effects of Pratylenchus neglectus Populations on Single and Interplantings of Alfalfa Grasses

The invasion by three different Utah populations of Pratylenchus neglectus (UTI, UT2, UT3) was similar in single and interplantings of ''Lahontan'' alfalfa and ''Fairway'' crested wheatgrass at 24 ñ 3 °C. Population UT3 was more pathogenic than UT1 and UT2 on both alfalfa and crested wheatgrass. Inoculum density was positively correlated with an invasion by P. neglectus. Invasions by UT3 at all initial populations (Pi) exceeded that of UT1 and UT2 for both single and interplanted treatments. The greatest reductions in shoot and root weights of alfalfa and crested wheatgrass were at a Pi of 8 P. neglectus/cm³ soil. Pi was negatively correlated with alfalfa and crested wheatgrass shoot and root growth and nematode reproduction. The reproductive factor (Rf) for UT3 exceeded that of UT1 and UT2 in single and interplantings at all inoculum levels. There were no differences in Rfin the Utah populations in single or interplantings. A nematode invasion increased with temperature and was greatest at 30 °C. Population UT3 was more pathogenic than UT1 and UT2 and reduced shoot and root growth at all soil temperatures. Populations UT1 and UT2 reduced shoot and root growth at 20-30 °C. Soil temperature was negatively correlated with shoot and root growth and positively correlated with nematode reproduction. Reproduction of UT3 exceeded that of UT1 and UT2 at all soil temperatures.     

Interactions Among Selected Endoparasitic Nematodes and Three Pseudomonads on Alfalfa

Meloidogyne hapla, Pratylenchus penetrans, and Helicotylenchus dihystera, reduced the growth of ''Saranac AR alfalfa seedlings when applied at concentrations of 50 nematodes per plant. All except P. penetrans reduced seedling growth when applied at 25 per seedling. M. hapla reduced growth when applied at 12 per seedling. Nematodes interacted with three pseudomonads to produce greater growth reductions than were obtained with single pathogens, suggesting synergistic relationships. Ditylenchus dipsaci, applied at 25 or 50 nematodes per seedling, reduced plant weight compared with weights of control plants, but did not interact with test bacteria. All of the nematodes except D. dipsaci produced root wounds which were invaded by bacteria.     

Fine Structure Analyses of Stem Nematode-Induced White Flagging in Medicago sativa

White flagging of alfalfa, Medicago sativa ''Ranger, found associated with Ditylenchus dipsaci in the Columbia River Basin was observed in northern Utah during 1971. This is a report on chloroplast changes, induced by D. dipsaci in alfalfa leaves, as observed with an electron microscope. Leaves from alfalfa plants infected with D. dipsaci were either devoid of normal pigmentation or displayed various shades of yellow-green. Cells of leaf tissue from noninfected plants exhibited normal chloroplast structure. By contrast, the chloroplast structure in cells of leaf tissue from infected plants showed progressive degradation as normal pigmentation decreased.     

Control of Pratylenchus penetrans and Meloidogyne hapla and Yield Response of Alfalfa Due to Oxamyl Seed Treatments

Alfalfa (Medicago sativa L. cv. Saranac) seed were soaked for 20 minutes in water, acetone, or methanol containing 10 or 50 mg/ml of oxamyl (Vydate L) or coated with a 2% aqueous cellulose solution containing the same amounts of oxamyl. Seed were analyzed for oxamyl by HPLC immediately after treatment and after 9 and 26 months of storage. Oxamyl content of alfalfa seed did not decline after 26 months of storage. The effects of seed treatment on growth of alfalfa and nematode control were examined using soils infested with Pratylenchus penetrans and Meloidogyne hapla. Germination was not affected by any of the seed treatments. Twenty-one days after sowing, the total growth of alfalfa seedlings grown from seed treated with 50 mg/ml of oxamyl in P. penetrans-infested soils had increased by 62% over controls. Nodulation per pot increased by as much as 267%, and the densities of P. penetrans per gram of root were reduced by as much as 73% compared to control plants. In M. hapla-infested soils, increases in plant growth (32%) and nodulation (71%) also occurred with oxamyl-treated seeds. Root gall reduction (86%) was also substantial due to oxamyl seed treatment.     

Effect of Ditylenchus dipsaci and Pratylenchus penetrans on Verticillium Wilt of Alfalfa

Verticillium albo-atrum wilt symptoms appeared faster and were significantly more severe in the presence of Ditylenchus dipsaci in Vernal, a wilt-susceptible cultivar, than in Marls Kabul, a wilt-resistant cultivar. Winter kill in the field was not affected by the nematode during the first winter, but 50% of plants were killed in the second winter. Forage yield from nematode-infected plants was significantly reduced the second year. Interaction with V. albo-atrum did not significantly reduce forage yields below that of D. dipsaci alone. Pratylenchus penetrans did not increase the severity of wilt symptoms in the presence of V. albo-atrum, nor did it affect forage yield in the greenhouse. It did, however, reduce alfalfa yields in presence of V. albo-atrum under field conditions. D. dipsaci and P. penetrans reproduced faster in Vernal than in Maris Kabul when the fungus was present.