Structural Changes Associated with Resistance of Soybean to Heterodera glycines

Subcellular responses to infection by Race 3 of Heterodera glycines in susceptible (''Lee'') and resistant (''Forrest'' and ''Bedford'') soybean cultivars were compared. Syncytial formation, initiated in susceptible as well as resistant soybean cultivars, was characterized by wall perforations, dense cytoplasm, and increased endoplasmic reticulum, In susceptible plants, syncytia developed continuously until nematode maturity. This included hypertrophy of nuclei, increase of rough endoplasmic reticulum in early stages of infection, and formation of wall ingrowths at a late stage of infection. In the resistant reaction in Forrest, a necrotic layer surrounded syncytium component cells demarcating them from surrounding normal cells and leading to syncytial necrosis. Wall appositions were prominently formed near the necrotic layer, and the cytoplasm of the syncytium component cells was extremely condensed. The whole syncytium became necrotic at a late stage of infection. Bedford had nuclear degeneration prior to cytoplasmic degradation. Chromatin was often scattered throughout the syncytial cytoplasm. Finally the whole syncytium became degenerated with plasmalemma completely detached from the syncytial cell walls. The differences in resistant responses reflect a difference in genetic composition of the soybean cultivars tested.     

Penetration and Development of Meloidogyne hapla in Resistant and Susceptible Alfalfa Under Differing Temperatures

Studies were conducted to examine under differing temperatures (12, 16, 20, 24, 28, and 32 C) the penetration anti development of Meloidogyne hapla in resistant lines ''298'' and ''Nev. Syn XX'', and susceptible ''Lahontan'' and ''Ranger'' hardy-type alfalfas. The results indicated that resistance to M. hapla was similar to that previously described for M. incognita in nonhardy alfalfa. Although initial penetration in resistant seedlings was similar to that of susceptible seedlings, nematode larvae failed to establish and develop in root tissues and nematode numbers subsequently declined. In susceptible seedlings, nematode development proceeded rapidly, and egg production began after 5 weeks. Temperature had little influence on the nematode development except to slow the response at the lower temperatures. Other studies were conducted to verify a previously reported immune (no penetration) reaction to M. hapla by the ''Vernal'' selection ''M-4''. When compared to the resistant (penetration without nematode development) Vernal selection ''M-9'' under differing temperatures (20, 24, 28, and 32 C), each selection was equally penetrated by M. hapla but at a lower level than in susceptible Ranger cuttings. Generally, no root galling was observed in either M-4 or M-9; however, very slight galling was found 35 days after inoculation on about 50% of these cuttings when grown at 32 C.     

Biological Relationship of Meloidogyne hapla Populations to Alfalfa Cultivars

Greenhouse and growth chamber studies were established to determine if there are pathological and physiological differences among Meloidogyne hapla populations from California (CA), Nevada (NV), Utah (UT), and Wyoming (WY) on alfalfa cultivars classified as resistant or susceptible to root-knot nematodes. In the greenhouse, plant survival was not consistent with resistance classifications. While all highly resistant Nevada Synthetic germplasm (Nev Syn XX) plants survived inoculation with all nematode populations, two cultivars classified as moderately resistant (''Chief'' and ''Kingstar'') survived (P ≤ 0.05) inoculation with M. hapla populations better than did ''Lobo'' cultivar, which is classified as resistant. Plant growth of Nev Syn XX was suppressed by only the CA population, whereas growth of the other alfalfa cultivars classified as M. hapla resistant or moderately resistant was suppressed by all nematode populations. Excluding Nev Syn XX, all alfalfa cultivars were severely galled and susceptible to all nematode populations. Except for Nev Syn XX, reproduction did not differ among the nematode populations on alfalfa cultivars. Nev Syn XX was not as favorable a host to CA as were the other cultivars; but, it was a good host (reproductive factor [Rf] = 37). Temperature affected plant resistance; the UT and WY populations were more pathogenic at 15-25 C, and CA was more pathogenic at 30 C. Nev Syn XX was susceptible to all nematode populations, except for CA, at only 30 C, and all other alfalfa cultivars were susceptible to all nematode populations at all temperatures.     

Symptomatology and Histopathology of Soybean Roots Infected by Pratylenchus scribneri and P. alleni

Size of lesions caused by Pratylenchus scribneri on roots of ''Clark 63'' soybean was correlated with nematode colony size within roots. A single nematode was capable of causing a detectable lesion. When a root became highly necrotic and shrunken, few nematodes but numerous eggs remained in the tissue. In histological sections made 5, 11, 18, and 45 d after planting, P. scribneri was located entirely within the cortex and generally was oriented longitudinally to the vascular cylinder, either outstretched in the same plane or coiled through several cells. Nematodes moved intracellularly, causing extensive rupturing of cell walls, retraction and disappearance of cytoplasm, and thickening of cell walls and necrosis of cells around feeding sites. Depth of penetration within the cortex and necrosis of cells increased with time after infection, eventually resulting in formation of cavities in the cortex and occasional secondary injury to the endodermis. Stele tissue was unaffected by feeding, and damage to the epidermis was limited to nematode entry points. Orientation of P. alleni and histopathology of its infection at 45 days were identical to those of P. scribneri, except that there was no injury to the endodermis.     

In Vitro Culture and Feeding Behavior of Belonolaimus longicaudatus on Excised Zea mays Roots

A greenhouse population of the sting nematode, Belonolaimus longicaudatus, obtained from an infested golf course in California''s Coachella Valley, was surface-decontaminated and cuhured on excised roots of Zea mays supported by Gamborg''s B5 medium. At 26-27 °C the females laid eggs, and newly emerged juveniles of the second generation completed three molts within 29 days after egg deposition. Sixty days after inoculation with 60 females and 40 males, an average of 529 nematodes and 83 eggs were recovered from the culture. The feeding process consisted of probing, stylet penetration, ingestion, and stylet retraction. Feeding seemed to be necessary before egg deposition or molting occurred. The sting nematode was observed feeding exclusively as an ectoparasite and preferably at the region of cell division and elongation. Vigorous feeding by many nematodes usually caused discoloration of root tips and termination of growth.     

Interaction of Vesicular-Arbuscular Mycorrhizae and Cultivars of Alfalfa Susceptible and Resistant to Meloidogyne hapla

The interaction between vesicular-arbuscular mycorrhizal (VAM) fungi and the root-knot nematode (Meloidogyne hapla) was investigated using both nematode-susceptible (Grasslands Wairau) and nematode-resistant (Nevada Synthetic XX) cultivars of alfalfa (Medicago sativa) at four levels of applied phosphate. Mycorrhizal inoculation improved plant growth and reduced nematode numbers and adult development in roots in dually infected cultures of the susceptible cultivar. The tolerance of plants to nematode infection and development when preinfected with mycorrhizal fungi was no greater than when they were inoculated with nematodes and mycorrhizal fungi simultaneously. Growth of plants of the resistant cultivar was unaffected by nematode inoculation but was improved by mycorrhizal inoculation. Numbers of nematode juveniles were lower in the roots of the resistant than of the susceptible cultivar and were further reduced by mycorrhizal inoculation, although no adult nematodes developed in any resistant cultivar treatment. Inoculation of alfalfa with VAM fungi increased the tolerance and resistance of a cultivar susceptible to M. hapla and improved the resistance of a resistant cultivar.     

Induction of Isoperoxidases in Resistant and Susceptible Tomato Cultivars by Meloidogyne incognita

Isoperoxidases were detected in resistant Rossol and susceptible Roma VF tomato roots uninfected and infected by Meloidogyne incognita. Syringaldazine, guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), and indoleacetic acid (IAA) were used as substrates, and the corresponding peroxidative activities were detected either in cytoplasmic or in cell wall fractions, except for IAA oxidase, which was measured in soluble and microsomal fractions. Isoperoxidase activities and cellular locations were induced differently in resistant and susceptible cultivars by nematodes. Nematode infestation markedly enhanced syringaldazine oxidase activity in cell walls of the resistant cultivar. This isoperoxidase is involved in the last step of lignin deposition in plants. Conversely, the susceptible cultivar reacted to M. incognita infection with an increase in cytoplasmic PPD-PC oxidase activity, which presumedly is involved in ethylene production; no changes in cell wall isoperoxidases were observed. IAA oxidase was inhibited in susceptible plants after nematode inoculation, whereas in resistant plants this activity increased in the soluble fraction and decreased in the microsomal fraction.     

Effects of Meloidogyne incognita on Nitrogen Fixation in Soybean

The effects of a North Carolina population of Meloidogyne incognita on N₂ fixation on root-knot-susceptible ''Lee 68'' and moderately resistant ''Forrest'' soybean were evaluated 50, 75, I00, and 135 days after inoculation with nematodes. Nematodes stimulated N₂ fixation in Lee 68 by 50 days and in Forrest by 75 days. At all other intervals, N₂ fixation was either depressed or unaffected by nematodes. Additional observations indicate that the susceptibility of Lee 68 is associated with greater rates of penetration by larvae and more favorable responses of host tissues to nematodes than occur in Forrest. With time, however, the histological reactions of both hosts became less favorable for nematode development. Resistant or hypersensitive responses became common in Forrest by 75 days but not in Lee 68 until 90 days after inoculation. This population of M. incognita may stimulate N₂ fixation at a specific time interval and depress it at others; therefore, disease of susceptible soybeans caused by this nematode is probably not primarily due to a net loss of fixed nitrogen but to pathogenicity similar to that which occurs on nonlegume hosts.     

Effects of Resistance in White Clover (Trifolium repens) on Heterodera trifolii

Clones of two partially resistant and two susceptible white clover, Trifolium repens, genotypes were exposed to eggs of Heterodera trifolii and nematode development in stained roots measured at 2, 4, 7, 11, 18, 23, and 37 days after inoculation. The differences in development between nematode populations in resistant and susceptible genotypes showed that resistance operated after infection during feeding and development. At 7 days after inoculation, counts of second-stage juveniles did not differ between genotypes, whereas at 37 days more adults had developed in the susceptible than in the resistant genotypes. In a separate experiment, cysts hosted by susceptible genotypes were larger and contained more eggs than those on resistant genotypes so that the product of the values for cysts per plant and for eggs per cyst resulted in a more sensitive measure of resistance than from using cysts per plant alone.     

A gene expression analysis of syncytia laser microdissected from the roots of the <Emphasis Type="Italic">Glycine max</Emphasis> (soybean) genotype PI 548402 (Peking) undergoing a resistant reaction after infection by <Emphasis Type="Italic">Heterodera glycines</Emphasis> (soybean cyst nematode)

The syncytium is a nurse cell formed within the roots of Glycine max by the plant parasitic nematode Heterodera glycines. Its development and maintenance are essential for nematode survival. The syncytium appears to undergo two developmental phases during its maturation into a functional nurse cell. The first phase is a parasitism phase where the nematode establishes the molecular circuitry that during the second phase ensures a compatible interaction with the plant cell. The cytological features of syncytia undergoing susceptible or resistant reactions appear the same during the parasitism phase. Depending on the outcome of any defense response, the second phase is a period of syncytium maintenance (susceptible reaction) or failure (resistant reaction). In the analyses presented here, the localized gene expression occurring at the syncytium during the resistant reaction was studied. This was accomplished by isolating syncytial cells from Glycine max genotype Peking (PI 548402) by laser capture microdissection. Microarray analyses using the Affymetrix^® soybean GeneChip^® directly compared Peking syncytia undergoing a resistant reaction to those undergoing a susceptible reaction during the parasitism phase of the resistant reaction. Those analyses revealed lipoxygenase-9 and lipoxygenase-4 as the most highly induced genes in the resistant reaction. The analysis also identified induced levels of components of the phenylpropanoid pathway. These genes included phenylalanine ammonia lyase, chalcone isomerase, isoflavone reductase, cinnamoyl-CoA reductase and caffeic acid O-methyltransferase. The presence of induced levels of these genes implies the importance of jasmonic acid and phenylpropanoid signaling pathways locally at the site of the syncytium during the resistance phase of the resistant reaction. The analysis also identified highly induced levels of four S-adenosylmethionine synthetase genes, the EARLY-RESPONSIVE TO DEHYDRATION 2 gene and the 14-3-3 gene known as GENERAL REGULATORY FACTOR 2. Subsequent analyses studied microdissected syncytial cells at 3, 6 and 9 days post infection (dpi) during the course of the resistant reaction, resulting in the identification of signature gene expression profiles at each time point in a single G. max genotype, Peking.     

Post-Infection Development and Histopathology of Meloidogyne incognita in Resistant Cotton

The numbers of Meloidogyne incognita larvae which migrated from cotton roots declined over a 16-day period, but the difference in numbers migrating from resistant and susceptible cultivars was not significant. Larvae penetrated susceptible roots, matured, and reproduced within 14 days following inoculation, whereas nematode development in the resistant roots was greatly retarded. Three types of histological responses were observed in infected, resistant roots, and these correlated with the degree of nematode development. Some galls were examined which contained only fragments of nematodes; others contained no detectable traces of developing larvae. Formation of druses in galls, but not in healthy tissue, was noted in both cultivars 20 days after inoculation. Massive invasion of roots resulted in deep longitudinal fissures of root cortex.     

Resistance Genes in a 'Williams 82' x 'Hartwig' Soybean Cross to an Inbred Line of Heterodera glycines

The number of resistance genes in soybean to soybean cyst nematode (SCN) Heterodera glycines was estimated using progeny from a cross of ''Williams 82'' x ''Hartwig'' (derived from ''Forrest''³ x PI 437.654) screened with a fourth-generation inbred nematode line derived from a race 3 field population of SCN. Numbers of females developing on roots of inoculated seedlings were assigned to phenotype cells (resistant, susceptible, or segregating) using Ward''s minimum variance cluster analysis. The ratio obtained from screening 220 F₃ soybean families was not significantly different from a 1:8:7 (resistant:segregating:susceptible) ratio, suggesting a two-gene system for resistance. The ratio obtained from screening 183 F₂ plants was not significantly different from a 3:13 (resistant:susceptible) ratio, indicating both a dominant (Rhg) and a recessive (rhg) resistance gene.     

Effects of Temperature on Development of Heterodera glycines on Glycine max and Phaseolus vulgaris

Soybean cyst nematode resistant ''Fayette'' and susceptible ''Williams 79'' soybeans (Glycine max) and resistant ''WIS (RRR) 36'' and susceptible ''Eagle'' snap beans (Phaseolus vulgaris) were used in determining the effects of host and temperature on the development, female production, sex ratios, and host response to Heterodera glycines. Temperatures were maintained constant at 16, 20, 24, 28, and 32 C using water-filled tanks. The most rapid development and greatest female production occurred between 20 and 28 C. The equation DS = 5(10⁻⁶)x²y² - 3(10⁻⁴)x²y - 2.8(10⁻³)x² - 1.94(10⁻²)y² + 0.4288x + 1.0220y - 12.7185, where DS = developmental stage, X = time, and Y = temperature, predicted the developmental stage of the nematode and accounted for 84% of the variation. Male : female ratios did not differ within this range and were generally less than one. At all temperatures the resistant soybean produced the greatest number of necrotic responses to H. glycines infection, followed by the resistant snap bean. The susceptible soybean and snap bean produced the fewest necrotic responses.     

Modification of Resistance Expression of Phaseolus vulgaris to Meloidogyne incognita by Elevated Soil Temperatures

The effect of temperature on the reaction of susceptible (Canario Divex) and resistant (A 211) bean pure lines to Meloidogyne incognita was studied with soil temperature tanks housed in a growth chamber at 22 or 24 C. Soil temperature remained constant at 16, 22, 24, 26, 30, or 32 C in several trials. Bean line A 211 was resistant at 16 and 22 C but was susceptible at 24 C and above. Resistance to root-knot nematode reproduction was affected by a lower temperature (24 C) than was resistance to root galling (26 C) in A 211. Incubation of A 211 at 30 C for 3 and 16 days after inoculation with M. incognita resulted in a significant increase in nematode reproduction and root galling, respectively. The resistant reactions of A 211 to nematode reproduction and root galling were retained when inoculated plants were incubated at 21 C for a minimum of 16 and 23 days, respectively, prior to high temperature treatment.     

Partial Characterization of Cytosolic Superoxide Dismutase Activity in the Interaction of Meloidogyne incognita with Two Cultivars of Glycine max

The closely related soybean (Glycine max) cultivars Centennial and Pickett 71 were confirmed to be resistant and susceptible, respectively, to the root-knot nematode Meloidogryne incognita. Increases in superoxide dismutase (SOD) activity were detected in roots of both soybean cultivars 48 hours following inoculation. Superoxide dismutase activity increased in roots of the susceptible cultivar overall, but declined after 96 hours in roots of the resistant cultivar. The isoelectric points of SOD isolated from preparasitic and parasitic developmental stages of the nematode appeared to differ. The SOD activity increased dramatically as nematodes matured and enlarged. Plant and nematode SOD were present as ca. 40-kDa cuprozinc dimers. Initial increases in SOD activity in infected tissue appeared to involve nematode regulation of plant gene expression. However, as the nematode enlarged, SOD activity could be detected within the female body only.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

Histopathology of Selected cultivars of Tobacco infected with Meloidogyne species

Rates of nematode penetration and the histopathology of root infections in fluecured tobacco cultivars ''McNair-944,'' ''Speight G-28,'' and ''NC-89'' with either Meloidogyne arenaria, M. incognita, M. hapla, or M. javanica were investigated. Penetration of root tips by juveniles of all species into the M. incognita-resistant NC-89 and G-28 was much less than that on the susceptible McNair-944. Few juveniles of M. incognita were detected in resistant cultivars 7 and 14 days after inoculation. Infection sites exhibited some cavities and extensive necrotic tissue at 14 days; less necrotic tissue and no intact nematodes were observed 35 days after inoculation. Although some females of M. arenaria reached maturity and produced eggs, considerable necrosis was induced in the resistant cultivars. Meloidogyne hapla and M. javanica developed on all cultivars, but there was necrotic tissue at some infection sites in the resistant cultivars. The occurrence of single multistructured nuclei in the syncytia of most M. hapla infections differed from the numerous small nuclei found in syncytia caused by the other three species.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

Selection Against Heterodera glycines Males by Soybean Lines with Genes for Resistance

Soybeans with genes for resistance select against Heterodera glycines with the corresponding genes for avirulence. There may be a differential effect of sex with some specific gene interactions, which would influence the magnitude of gene frequency changes. No effect on H. glycines males was detected with one selected nematode population and the resistant soybean line PI88788. The selective effect of PI89772 against male nematodes was greater with two inbred nematode populations than with one selected (on PI88788) population, presumably due to differences in H. glycines gene frequencies. ''Peking'' also had few males with the one inbred nematode population, whereas Forrest and ''Pickett 71'' had intermediate numbers. Apparently Forrest and Pickett 71 did not get all the Peking genes for resistance that affect male as well as female nematode development. Other H. glycines-soybean genes stop only females, since there were few or no cysts, except on the susceptible soybean Williams. The number of males'' phenotype will help identify specific genes in both organisms.     

Postinfection Development of Rotylenchulus reniformis on Resistant Gossypium barbadense Accessions

The reniform nematode (Rotylenchulus reniformis) causes significant cotton (Gossypium hirsutum) losses in the southeastern United States. The research objective was to describe the effects of two resistant G. barbadense lines (cultivar TX 110 and accession GB 713) on development and fecundity of reniform nematode. Nematode development and fecundity were evaluated on the resistant lines and susceptible G. hirsutum cultivar Deltapine 16 in three repeated growth chamber experiments. Nematode development on roots early and late in the infection cycle was measured at set intervals from 1 to 25 d after inoculation (DAI) and genotypes were compared based on the number of nematodes in four developmental stages (vermiform, swelling, reniform, and gravid). At 15, 20, and 25 DAI, egg production by individual females parasitizing each genotype was measured. Unique reniform nematode developmental patterns were noted on each of the cotton genotypes. During the early stages of infection, infection and development occurred 1 d faster on susceptible cotton than on the resistant genotypes. Later, progression to the reniform and gravid stages of development occurred first on the susceptible genotype, followed by G. barbadense cultivar TX 110, and finally G. barbadense accession GB 713. Egg production by individual nematodes infecting the three genotypes was similar. This study corroborates delayed development previously reported on G. barbadense cultivar TX 110 and is the first report of delayed infection and development associated with G. barbadense accession GB 713. The different developmental patterns in the resistant genotypes suggest that unique or additional loci may confer resistance in these two lines.