Penetration and Development of Meloidogyne arenaria on Two New Grape Rootstocks

Penetration, development, and reproduction of a virulent ''Harmony'' population of Meloidogyne arenaria was studied on two nematode-resistant grape rootstocks 10-17A and 6-19B. ''Cabernet Sauvignon'' was used as a susceptible control for comparison. Plants were inoculated with 100 freshly hatched second-stage juveniles (J2) of M. arenaria. Greater numbers of J2 penetrated roots of ''Cabernet'' than 10-17A, and none penetrated roots of 6-19B 4 days after inoculation (DAI). At 7 DAI, vermiform J2 advanced to sausage-shaped J2 in roots of ''Cabernet,'' penetrated roots of 6-19B, and had egressed from roots of 10-17A. Resistant rootstocks expressed hypersensitive responses to penetrating J2 along the root epidermis, among the cortical cells, and along the differentiating vascular bundles. At 13 DAI, 68% of the J2 had attained globose stage in roots of ''Cabernet,'' whereas there was no development of vermiform J2 in roots of the other two rootstocks. The nematodes reproduced only in roots of ''Cabernet.'' Lack of development of J2 in roots of the two resistant grape rootstocks might be the result of a hypersensitive response to J2 feeding.     

Reactions of Grape Rootstocks to Pratylenchus vulnus and Meloidogyne spp.

Five grape rootstocks were inoculated with 0, 100, 1,000, and 10,000 Pratylenchus vulnus. Dogridge and Saltcreek supported low average total numbers of P. vulnus, 136-705/pot, at 12 months after inoculation. Growth of both rootstocks was not affected. Harmony, Couderc 1613, and Ganzin 1 supported high average total numbers, 6-856 times the inoculum levels. Numbers in Harmony continued to increase at all levels but reduced root weight only at the 10,000 level after 12 months. Numbers in Couderc 1613 decreased by 15-30% after 12 months, and root weight was reduced at the 10,000 level. In Ganzin 1, total nematode numbers diminished after 12 months but were still at high levels; growth reduction was proportional to numbers of nematodes added. Meloidogyne incognita, M. javanica, and M. arenaria produced galls and egg masses in Harmony and Couderc 1613 only at 36 C. Galling in Ganzin 1 increased with increasing temperature. Galls in Ganzin 1 at 18 C supported mature females after 90 days. Harmony was resistant to M. incognita in single and concomitant inoculations of P. vulnus and M. incognita. At 250 days after inoculation, total numbers of P. vulnus increased above the inoculum level and the 150-day values; increase was greatest in P. vulnus added singly. Neither nematode species affected growth of Harmony.     

Variability among Populations of Meloidogyne arenaria

Variability in reproduction and pathogenicity of 12 populations of Meloidogyne arenaria race 1 was evaluated on Florunner peanut, Centennial soybean, Rutgers tomato, G70, K326, and Mc944 tobacco, and Carolina Cayenne, Mississippi Nemaheart, and Santanka pepper. Differences among M. arenaria populations in rates of egg production 45 days after inoculation were observed for all cultivars except Santanka pepper. Differences among populations in dry top weights or fresh root weights were recorded on all cultivars. Numbers of nematode eggs produced on Florunner peanut varied from 3,419 to 11,593/g fresh root weight. On resistant tobacco cultivars (G70 and K326), one nematode population produced high numbers of eggs (12,042 and 6,499/g fresh root weight on G70 and K326, respectively), whereas the other populations produced low numbers of eggs (less than 500 eggs/g fresh root weight on both cultivars). Two variant M. arenaria race 1 populations were identified by factor analysis of reproductive rates on all nine cultivars. Differences m reproduction and pathogenicity observed among populations would affect the design of sustainable management systems for M. arenaria.     

A Novel Technique for Infesting Field Sites with Encapsulated Eggs of Meloidogyne spp.

Eggs of Meloidogyne arenaria race 1 were encapsulated in calcium alginate for use as inoculum to infest peanut field plots. Some eggs within the capsules remained viable up to 10 weeks after preparation. A field site was successfully infested at peanut planting and (or) 6 weeks later. Dual applications of nematode inoculum (at planting and 6 weeks later) were superior to single applications (at planting or 6 weeks after planting). Field-site infestation levels at the end of the first year were related to the amount of inoculum dispersed and timing of the infestation (P = 0.001). Peanut yield was only slightly affected in the first year, but significant (P = 0.02) yield suppression occurred during the second year after field infestations. The negative relationship between the numbers of M. arenaria eggs and juveniles per 500 cm³ soil in the fall and the percentage of peanut hull galled the second year was described by a quadratic model (P = 0.002, R² = 0.41).     

Resistance of Interspecific Arachis Breeding Lines to Meloidogyne javanica and an Undescribed Meloidogyne Species

Resistance to a peanut-parasitic population of Meloidogyne javanica and an undescribed Meloidogyne sp. in peanut breeding lines selected for resistance to Meloidogyne javanica was examined in greenhouse tests. The interspecific hybrid TxAG-7 was resistant to reproduction of Meloidogyne javanica, M. javanica, and Meloidogyne sp. An Meloidogyne javanica-resistant selection from the second backcross (BC) of TxAG-7 to the susceptible cultivar Florunner also was resistant to M. javanica but appeared to be segregating for resistance to the Meloidogyne sp. When reproduction of M. javanica and Meloidogyne javanica were compared on five BC4F3 peanut breeding lines, each derived from Meloidogyne javanica-susceptible BC4F2 individuals, all five lines segregated for resistance to M. javanica, whereas four of the lines appeared to be susceptible to Meloidogyne javanica. These data indicate that several peanut lines selected for resistance to Meloidogyne javanica also contain genes for resistance to populations of M. javanica and the undescribed Meloidogyne sp. that are parasitic on peanut. Further, differences in segregation patterns suggest that resistance to each Meloidogyne sp. is conditioned by different genes.     

Variability in Reproduction of Four Populations of Meloidogyne incognita on Six Cultivars of Cotton

A range of virulence levels was found in four populations of Meloidogyne incognita collected from cotton fields of the Punjab region of Pakistan. The most virulent population was associated with development of larger gall size, larger giant cell formation and improved success of juveniles transitioning into reproducing adults. The most virulent nematode population, MI-78, emanated from cotton cultivar NIAB-78. This cotton cultivar also possessed the greatest level of resistance to the three other nematode populations evaluated in this study. The source of plant resistance was not evident during root penetration by second-stage juveniles (J2), but became apparent as nematode feeding was attempted. Although one other cotton cultivar, CIM-506, could also be designated as showing a level of resistance, none of the other cultivars reduced any nematode stage by more than 75% of that achieved on the best host. These data provide an example of a single cotton cultivar that could have short-term utility in field settings. The data also provide insight for future cotton breeding programs.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Damage Functions for Three Meloidogyne Species on Arachis hypogaea in Texas

The yield response of Florunner peanut to different initial population (Pi) densities of Meloidogyne arenaria, M. javanica, and an undescribed Meloidogyne species (isolate 93-13a) was determined in microplots in 1995 and 1996. Seven Pi''s (0, 0.5, 1, 5, 10, 50, and 100 eggs and J2/500 cm³ soil) were used for each Meloidogyne species in both years. The three species reproduced abundantly on Florunner in both years. In 1995, mean reproduction differed among the three species; mean Rf values were 10,253 for isolate 93-13, 4,256 for M. arenaria, and 513 for M. javanica. In 1996, the reproduction of M. arenaria (mean Rf = 7,820) and isolate 93-13a (mean Rf = 7,506) were similar, and both had greater reproduction on peanut than did M. javanica (mean Rf = 2,325). All three nematode species caused root and pod galling, and a positive relationship was observed between Pi and the percentage of pods galled. Meloidogyne arenaria caused a higher percentage of pod galling than did M. javanica or isolate 93-13a. A negative linear relationship between log₁₀ (Pi + 1) and pod yield was observed for all three nematode species each year. The yield response slopes were similar except for that of M. javanica, which was less negative than that of isolate 93-13a in 1995, and less negative than that of M. arenaria and isolate 93-13a in 1996.     

Resistance in Lycopersicon peruvianum to Isolates of Mi Gene-Compatible Meloidogyne Populations

Root-knot nematode resistance of F₁ progeny of an intraspecific hybrid (Lycopersicon peruvianum var. glandulosum Acc. No. 126443 x L. peruvianum Acc. No. 270435), L. esculentum cv. Piersol (possessing resistance gene Mi), and L. esculentum cv. St. Pierre (susceptible) was compared. Resistance to 1) isolates of two Meloidogyne incognita populations artificially selected for parasitism on tomato plants possessing the Mi gene, 2) the wild type parent populations, 3) four naturally occurring resistance (Mi gene)-breaking populations of M. incognita, M. arenaria, and two undesignated Meloidogyne spp., and 4) a population of M. hapla was indexed by numbers of egg masses produced on root systems in a greenhouse experiment. Artificially selected M. incognita isolates reproduced abundantly on Piersol, but not (P = 0.01) on resistant F₁ hybrids. Thus, the gene(s) for resistance in the F₁ hybrid differs from the Mi gene in Piersol. Four naturally occurring resistance-breaking populations reproduced extensively on Piersol and on the F₁ hybrid, demonstrating ability to circumvent both types of resistance. Meloidogyne hapla reproduced on F₁ hybrid plants, but at significantly (P = 0.01) lower levels than on Piersol.     

Effect of Carbamate,Organophosphate, and Avermectin Nematicides on Oxygen Consumption by Three Meloidogyne spp.

Second-stage juveniles (I2) of Meloidogyne arenaria consumed more oxygen (P ≤ 0.05) than M. incognita J2, which in turn consumed more than M. javanica J2 (4,820, 4,530, and 3,970 μl per hour per g nematode dryweight, respectively). Decrease in oxygen consumption depended on the nematicide used. Except for aldicarb, there was no differential sensitivity among the three nematode species. Meloidogyne javanica had a greater percentage decrease (P ≤ 0.05) in oxygen uptake when treated with aldicarb, relative to the untreated control, than either M. arenaria or M. incognita. Meloidogyne javanica J2 had a greater degree of recovery from fenamiphos or aldicarb intoxication, after subsequent transfer to water, than did M. incognita. This finding may relate to differential sensitivity among Meloidogyne spp. in the field. Degree of respiratory inhibition and loss of nematode motility for M. javanica after exposure to the nematicides were positively correlated (P ≤ 0.05).     

Relative Damage Functions and Reproductive Potentials of Meloidogyne arenaria and M. hapla on Peanut

The reproductive potential and damage functions for Meloidogyne hapla and M. arenaria race 1 on Virginia-type peanuts (Arachis hypogaea cv. Florigiant) were determined over 2 years in microplot experiments in North Carolina. Peanut yield suppression and damage to pods as a result of galling were greatest in response to M. arenaria (P = 0.01). Damage functions for the two species were adequately described by the quadratic models: yield (g/plot) = 398 - 17.1 (log₁₀[Pi + 1]) - 17.0(log₁₀[Pi + 1])²; (R² = 0.83, P = 0.0001) for M. arenaria; and yield = 388 - 10.2(log₁₀[Pi + 1]) - 7.5(log₁₀[Pi + 1])², (R² = 0.30, P = 0.0001) for M. hapla. Both species caused galling on pods, but this was more severe in response to M. arenaria. Reproduction of M. arenaria race 1 was greater than M. hapla on peanut, which accounts in part for the more severe pod galling. Peanut was an excellent host for both M. arenaria race 1 and for M. hapla, but reproduction by M. hapla was more variable.     

Reproduction of Meloidogyne spp. on Resistant Peanut Genotypes from Three Breeding Programs

Three described species of root-knot nematode parasitize peanut (Arachis hypogaea): Meloidogyne arenaria race 1 (Ma), M. hapla (Mh), and M. javanica (Mj). Peanut cultivars with broad resistance to Meloidogyne spp. will be useful regardless of the species present in the field. The objective of this study was to determine whether peanut genotypes with resistance to M. arenaria originating from three different breeding programs were also resistant to M. hapla and M. javanica. The experiment used a factorial arrangement (completely randomized) with peanut genotype and nematode population as the factors. The five peanut genotypes were ''COAN'' and AT 0812 (highly resistant to Ma), C209-6-13 (moderately resistant to Ma), and ''Southern Runner'' and ''Georgia Green'' (susceptible to Ma). The four nematode populations were two isolates of Ma (Gibbs and Gop) and one isolate each of Mh and Mj. On COAN or AT 0812, both Ma and Mj produced <10% of the eggs produced on Georgia Green. On the peanut genotype C209-6-13, Ma and Mj produced about 50% of the eggs produced on Georgia Green. None of the resistant genotypes exhibited a high level of resistance to Mh. The lack of resistance to Mh in any cultivars or advanced germplasm is a concern because the identity of a Meloidogyne sp. in a particular peanut field is generally not known. Breeding efforts should focus on moving genes for resistance to M. hapla into advanced peanut germplasm, and combining genes for resistance to the major Meloidogyne spp. in a single cultivar.     

Host Status of Endophyte-Infected and Noninfected Tall Fescue Grass to Meloidogyne spp

Tall fescue grass cultivars with or without endophytes were evaluated for their susceptibility to Meloidogyne incognita in the greenhouse. Tall fescue cultivars evaluated included, i) wild-type Jesup (E+, ergot-producing endophyte present), ii) endophyte-free Jesup (E-, no endophyte present), iii) Jesup (Max-Q, non-ergot producing endophyte) and iv) Georgia 5 (E+). Peach was included as the control. Peach supported greater (P ≤ 0.05) reproduction of M. incognita than all tall fescue cultivars. Differences in reproduction were not detected among the tall fescue cultivars and all cultivars were rated as either poor or nonhosts for M. incognita. Suppression of M. incognita reproduction was not influenced by endophyte status. In two other greenhouse experiments, host susceptibility of tall fescue grasses to two M. incognita isolates (BY-peach isolate and GA-peach isolate) did not appear to be related to fungal endophyte strain [i.e., Jesup (Max-Q; nontoxic endophyte strain) vs. Bulldog 51 (toxic endophyte strain)]. Host status of tall fescue varied with species of root-knot nematode. Jesup (Max-Q) was rated as a nonhost for M. incognita (BY-peach isolate and GA-peach isolate) and M. hapla, a poor host for M. javanica and a good host for M. arenaria. Bulldog 51 tall fescue was also a good host for M. arenaria and M. javanica, but not M. incognita. Jesup (Max-Q) tall fescue may have potential as a preplant control strategy for M. incognita and M. hapla in southeastern and northeastern United States, respectively.     

Effects of Meloidogyne spp. and Rhizoctonia solani on the Growth of Grapevine Rootings

A disease complex involving Meloidogyne incognita and Rhizoctonia solani was associated with stunting of grapevines in a field nursery. Nematode reproduction was occurring on both susceptible and resistant cultivars, and pot experiments were conducted to determine the virulence of this M. incognita population, and of M. javanica and M. hapla populations, to V. vinifera cv. Colombard (susceptible) and to V. champinii cv. Ramsey (regarded locally as highly resistant). The virulence of R. solani isolates obtained from roots of diseased grapevines also was determined both alone and in combination with M. incognita. Ramsey was susceptible to M. incognita (reproduction ratio 9.8 to 18.4 in a shadehouse and heated glasshouse, respectively) but was resistant to M. javanica and M. hapla. Colombard was susceptible to M. incognita (reproduction ratio 24.3 and 41.3, respectively) and M. javanica. Shoot growth was suppressed (by 35%) by M. incognita and, to a lesser extent, by M. hapla. Colombard roots were more severely galled than Ramsey roots by all three species, and nematode reproduction was higher on Colombard. Isolates of R. solani assigned to putative anastomosis groups 2-1 and 4, and an unidentified isolate, colonized and induced rotting of grapevine roots. Ramsey was more susceptible to root rotting than Colombard. Shoot growth was inhibited by up to 15% by several AG 4 isolates and by 20% by the AG 2-1 isolate. AG 4 isolates varied in their virulence. Root rotting was higher when grapevines were inoculated with both M. incognita and R. solani and was highest when nematode inoculation preceded the fungus. Shoot weights were lower when vines were inoculated with the nematode 13 days before the fungus compared with inoculation with both the nematode and the fungus on the same day. It was concluded that both the M. incognita population and some R. solani isolates were virulent against both Colombard and Ramsey, and that measures to prevent spread in nursery stock were therefore important.     

Reproduction of Mi-Virulent Meloidogyne incognita Isolates on Lycopersicon spp.

Selection of detectable numbers of Mi-virulent root-knot nematodes has necessitated a greater understanding of nematode responses to new sources of resistance. During the course of this research, we compared the reproduction of four geographically distinct Mi-virulent root-knot nematode isolates on three resistant accessions of Lycopersicon peruvianum. Each accession carried a different resistant gene, Mi-3, Mi-7, or Mi-8. All nematode isolates were verified as Meloidogyne incognita using diagnostic markers in the mitochondrial genome of the nematode. Reproduction of Mi-virulent isolates W1, 133 and HM, measured as eggs per g of root, was greatest on the Mi-7 carrying accession and least on the Mi-8 carrying accession. In general, Mi-3 behaved similar to the Mi-8 carrying accession. Reproduction of the four nematode isolates was also compared on both Mi and non-Mi-carrying L. esculentum cultivars and a susceptible L. peruvianum accession. Resistance mediated by Mi in L. esculentum still impacted the Mi-virulent nematodes with fewer eggs per g of root on the resistant cultivar (P ≤ 0.05). Preliminary histological studies suggests that Mi-8 resistance is mediated by a hypersensitive response, similar to Mi.     

Greenhouse Studies on the Effect of Marigolds (Tagetes spp.) on Four Meloidogyne Species

The effects of preplanted marigold on tomato root galling and multiplication of Meloidogyne incognita, M. javanica, M. arenaria, and M. hapla were studied. Marigold cultivars of Tagetes patula, T. erecta, T. signata, and a Tagetes hybrid all reduced galling and numbers of second-stage juveniles in subsequent tomato compared to the tomato-tomato control. All four Meloidogyne spp. reproduced on T. signata ''Tangerine Gem''. Several cultivars of T. patula and T. erecta suppressed galling and reproduction of Meloidogyne spp. on tomato to levels lower than or comparable to a fallow control. Phytotoxic effects of marigold on tomato were not observed. Several of the tested marigold cultivars are ready for full-scale field evaluation against Meloidogyne spp.     

Sensitive PCR Detection of Meloidogyne arenaria, M. incognita, and M. javanica Extracted from Soil

We have developed a simple PCR assay protocol for detection of the root-knot nematode (RKN) species Meloidogyne arenaria, M. incognita, and M. javanica extracted from soil. Nematodes are extracted from soil using Baermann funnels and centrifugal flotation. The nematode-containing fraction is then digested with proteinase K, and a PCR assay is carried out with primers specific for this group of RKN and with universal primers spanning the ITS of rRNA genes. The presence of RKN J2 can be detected among large numbers of other plant-parasitic and free-living nematodes. The procedure was tested with several soil types and crops from different locations and was found to be sensitive and accurate. Analysis of unknowns and spiked soil samples indicated that detection sensitivity was the same as or higher than by microscopic examination.