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Published online 19 March 2008
Published in Crop Sci 48:553-561 (2008)
© 2008 Crop Science Society of America
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Genetic Determinants of Differential Resistance to Root-Knot Nematode Reproduction and Galling in Lima Bean

P. A. Robertsa,*, W. C. Matthewsa, J. D. Ehlersb and D. Helmsc

a Dep. of Nematology, Univ. of California, Riverside, CA 92521
b Dep. of Botany and Plant Sciences, Univ. of California, Riverside, CA 92521
c Dep. of Agronomy and Range Science, Univ. of California, Davis, CA 95616. This research was supported in part by grants from the California Large and Baby Lima Councils and California Dry Bean Advisory Board

* Corresponding author (philip.roberts{at}ucr.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Root-knot nematodes (Meloidogyne spp.) are important root parasites that limit lima bean (Phaseolus lunatus L.) production, although genetic resistance is available. In accession L-136, we investigated the apparent independence of genes controlling nematode reproduction and root galling. Segregation of nematode egg production and root-galling phenotypes in F1, F2, F2:3, and F2:7-9 populations from L-136 x susceptible ‘Henderson Bush’ was determined in growth pouch, greenhouse pot, and field screenings. Three nuclear genes designated mir-1 (recessive), Mig-1 (dominant), and Mjg-1 (dominant) were found to control M. incognita reproduction, M. incognita galling, and M. javanica galling, respectively. Contrast analysis indicated that gene combinations affected nematode egg production and gene combinations provided comprehensive resistance. This independent resistance to root-knot galling and reproduction, reported for other leguminous crops and cotton (Gossypium spp.), has important implications for breeding broad-based nematode resistance.

Abbreviations: LSD, least significant difference • RIL, recombinant inbred line


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
ROOT-KNOT NEMATODES (Meloidogyne spp.) are important parasites of crop plants worldwide that have wide host ranges and cause significant crop losses (Sasser, 1977). Crop losses caused by root-knot in lima bean (Phaseolus lunatus L.) have been recognized for more than a half-century and have resulted in efforts to breed cultivars with nematode resistance (Helms et al., 2004). Resistance to root-knot in lima bean was identified in germplasm and derived breeding lines by Allard and colleagues in the 1950s (Allard, 1954; McGuire and Allard, 1958). Although a formal genetic analysis of the resistance was not made at that time, field screenings of nematode-induced root-galling symptoms in segregating populations in the breeding program indicated that resistance derived from accession L-136 was controlled by multiple genes that were dominant (McGuire et al., 1961). Additional sources of resistance in other P. lunatus accessions and lines including ‘L-77’ and ‘L-76’ were used in the California breeding program to develop nematode-resistant large-seeded lima bean cultivars, including White Ventura N, Maria, and UC 92 (Tucker, 1969; California Crop Improvement Association, 1979; Temple and Helms, 1992). However, genetic analysis of the root-knot nematode resistance in these sources has not been reported. Recently, we used the L-136 source of nematode resistance to develop the first nematode-resistant small-seeded or "baby" lima bean cultivar, Cariblanco N (Helms et al., 2004).

Resistance in L-136 and the derived Cariblanco N was found to be effective against both Meloidogyne incognita (Kofoid and White) Chitwood and M. javanica (Treub) Chitwood, the most common root-knot species in California lima production areas (Helms et al., 2004). The resistance derived from L-77 and L-76 and bred into the large-seeded cultivars was shown to be effective only against M. incognita (Roberts and Matthews, unpublished data). Detailed screening of L-136, Cariblanco N, and derived progenies revealed that the resistance to M. incognita suppressed both nematode reproduction and nematode-induced root-galling reaction. In contrast, the resistance to M. javanica was found to be effective in suppressing root-galling but not nematode reproduction. The differences in the specificity of the resistance phenotypes in L-136 presented challenges in the development of Cariblanco N because the different traits were found to segregate in progenies developed for selection of resistance combined with desirable agronomic traits. Therefore, coscreening progenies for the three traits (resistance to M. incognita reproduction, resistance to M. incognita galling, resistance to M. javanica galling) was necessary because each trait appeared to segregate independently, and the goal of the breeding effort was to introgress the full complement of root-knot nematode resistance from L-136 (Helms et al., 2004).

In most host plants, root-knot nematodes reproduce and induce root galling in compatible interactions, and major resistance genes suppress both root galling and nematode reproduction (Roberts, 1995). Thus, the differential plant response to nematode root galling and reproduction that we observed when breeding the L-136 resistance into lima bean cultivar Cariblanco N is uncommon, although it also has been observed in two other leguminous plants, common bean (Phaseolus vulgaris L.) (Fassuliotis et al., 1970) and soybean [Glycine max (L.) Merr.] (Harris et al., 2003), and in cotton (Gossypium spp.) (Shepherd, 1979). However, these observations on differential resistance reactions were not followed by a formal genetic analysis of the trait determinants involved in each case.

On the basis of these observations, a formal genetic analysis of the root-knot nematode resistance traits in L-136 was made to determine the contributions of each trait to the resistance phenotypes and to guide future breeding efforts in lima bean. The apparent independence of resistance effects on nematode reproduction and induced root galling is quite uncommon for root-knot nematode resistance gene action among various host plants, and a clearer definition of this system may provide useful genotypes for molecular analysis of resistance pathways. The specific objectives of this study were (i) to examine the inheritance relationships of the specific root-knot resistance traits in L-136, and (ii) to determine the resistance phenotype expressed by the genes singly and in different combinations.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Nematode Isolates
A Meloidogyne incognita race 3 isolate ‘Project 77’ collected from a cotton field near Tipton, Tulare County, CA, and maintained on susceptible tomato ‘UC82’ in greenhouse culture was used for all M. incognita resistance assays. An isolate (‘811’) of M. javanica collected from a cowpea [Vigna unguiculata (L.) Walp.] field near Chino, San Bernardino County, CA, and maintained on susceptible tomato UC82 in greenhouse culture was used for all M. javanica resistance assays. Species and race identity of these isolates was confirmed by isozyme phenotyping and by a host differential test as described previously (Roberts et al., 1996).

Plant Populations
Lima bean crosses were made between resistant L-136 and susceptible ‘Henderson Bush’. To ensure homozygosity, plants of the resistant parent were used for crossing only after several generations of inbreeding. Crosses were made in the morning by pollinating flowers that had been emasculated the previous afternoon with newly opened flowers of the male or pollen donor parent. Removal of unopened pollen sacs helped safeguard against selfing and increased the frequency of hybrids. Indeterminate growth habit, a dominant trait, was used as a morphological marker to verify hybrids. L-136 has an indeterminate (vine) growth habit and was used as the pollen donor in all crosses. Henderson Bush has a determinate (bush) growth habit and was used as the female parent in all crosses.

Hybrid (F1) plants with indeterminate growth habit were used for resistance screening and to produce F2 and F2:7–9 recombinant inbred line (RIL) populations. The F2:7 RIL population was developed through single seed descent. One hundred and twenty F2 plants were selfed and produced F3 seed. One F3 seed per family was selected randomly from a minimum of 100 seeds. This procedure was repeated at each generation. A preliminary F7 screen indicated some lines segregated for resistance. Those lines were advanced to the F9 generation and screened to determine uniformity in their resistance reactions, resulting in 119 RILs for resistance evaluation.

For additional inheritance testing of resistance to M. incognita reproduction, F2 and F2:3 populations were derived from crosses between Henderson Bush (female parent) and an indeterminate RIL characterized as resistant to M. incognita reproduction. This line reacted to M. incognita and M. javanica infection with significant galling of the primary root similar to that observed on the susceptible parent Henderson Bush.

Resistance Assays
Resistance assays were conducted with plants grown in pots in a greenhouse, in seed germination pouches, and in nematode-infested field plots. F1 and F2:7–9 RILs were screened in pots for nematode-induced root galling and nematode egg production. Pouches were used to evaluate F2 and F2:3 families where a precise measure of nematode reproduction was required. Two large F2 populations were tested under field conditions for root-galling reaction.

In separate experiments, F1 and F2:7–9 RILs were tested in pots with M. incognita and M. javanica. The F1 was replicated seven times, and each RIL was replicated four times. Plants were direct-seeded in 10-cm-diam. plastic pots filled with steam-sterilized sand and were inoculated when the first set of trifoliate leaves had expanded, about 3 wk after planting. Nematode egg inoculum was extracted from tomato roots with NaOCl (Hussey and Barker, 1973) and used immediately or within 1 wk of storage in water at 5°C. Seedlings were inoculated through a syringe with approximately 50,000 eggs in 10 mL of water divided between two places in the pot. The 7-cm-long syringe needle had three holes at 1.5-cm intervals to facilitate uniform inoculation with depth. After inoculation, plants were fertilized by surface application with 2.5 mL of 14-14-14 controlled release fertilizer (Scotts-Sierra Horticultural Products Co.). Pots were drip-irrigated as needed to maintain optimal growth. Air temperatures in the greenhouse were maintained between 27 and 35°C during the day and 24 ± 1°C at night. Soil temperatures were in the range of 24 to 27°C during the experiments.

Pot-tested plants were evaluated for resistance reaction 60 d after inoculation when root systems were rinsed free of soil. Resistance to nematode-induced root galling was scored as presence or absence of "primary galling," which was the large coalesced galling response visible on the primary root (Fig. 1 ). In addition, root systems were examined visually under a 10x magnifying lamp for presence or absence of nematode egg masses as a measure of plant resistance to nematode reproduction (Fig. 1). Resistance to reproduction was assessed further by extracting and enumerating nematode eggs from roots with NaOCl. Extracted eggs were expressed as total eggs per root system and number of eggs per gram of fresh root. All F1 plants and a subset of 19 indeterminate lines of the 119 RILs, with two or three lines representing each resistance genotype, were assayed for nematode egg production.


Figure 1
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  		Figure 1. Root-galling symptoms and egg mass production of (A–C) Meloidogyne incognita and (D) M. javanica 60 d after inoculation in pots on lima bean recombinant inbred lines from the cross of susceptible Henderson Bush x resistant L-136 possessing different combinations of resistance genes: (A) susceptible to both primary root galling and reproduction; (B) resistant to both primary root galling and reproduction; (C) susceptible to primary root galling and resistant to reproduction; (D) resistant to primary root galling and susceptible to reproduction. Egg masses are stained blue with erioglaucine.

 
One F2 family and 27 F2:3 families were tested for resistance to M. incognita reproduction in seed germination pouches (CYG pouch, Mega International, West St. Paul, MN) using the protocol of Ehlers et al. (2000) with minor modifications. Lima bean seeds were germinated in the pouches, and fungal contamination was minimized by removal of seed coats soon after germination. Pouches were placed in plastic file folders, two per folder, which were held in file crates. Pouches were moved to a controlled environment chamber maintained at 26.7 ± 0.6°C and 16 h daylength. After 14 d, plants were inoculated with 2000 freshly hatched nematode second-stage juveniles per pouch, prepared by extracting eggs from tomato roots with NaOCl and hatching them in tap water at 27°C. Pouches were positioned horizontally for 24 h after inoculation for uniform infection of roots. Plants were watered with half-strength Hoagland's solution (Hoagland and Arnon, 1950) until a response to the fertilizer was observed and were kept moist with water for the remainder of the test. Pouches were flooded with 75 mg L–1 erioglaucine (Sigma Chemical Co., St. Louis, MO) 30 d after inoculation to stain egg masses for counting. Pouch-tested F2 plants were classified resistant or susceptible on the basis of parental egg mass counts. Egg masses on the resistant parent L-136 ranged from 5 to 55 per plant; F2 plants with ≤55 were scored as resistant, and those with >55 were scored as susceptible.

Twenty-seven F2 plants were retained for further resistance evaluation and progeny development. Nine were selected from the resistant group, nine from the moderately susceptible group (56–125 egg masses per plant), and nine from the highly susceptible group (>125 egg masses per plant). These F2 plants were transferred from pouches to 15-cm-diam. pots containing steam-sterilized 80:20 sand:peat moss mix. After 60 d, F3 seed was collected and root systems were assayed for primary galling and nematode egg production.

Twelve plants per F2-derived F3 family were screened with M. incognita in pouches. The highest score (34 egg masses) in the range of the resistant parent L-136 determined resistance classification of F3 plants. F3 plants with ≤34 egg masses were classified resistant, and >34 as susceptible. Because of overlap in the parental egg mass counts, plants were also scored visually, under 10x magnification, for "secondary galling," which was localized galling on secondary roots. Secondary galling, restricted in size and distinguishable from the severe galling of the primary root (Fig. 1), was clearly visible on roots of the susceptible parent Henderson Bush but not on roots of resistant L-136. Evaluation for secondary galling helped to identify susceptible plants with low (≤34) and resistant plants with high (>34) egg mass numbers. Out of 327 F3 plants, 20 were classified by secondary galling reaction only.

In field tests, F2 populations were screened at the University of California Kearney Agricultural Research and Extension Center (Parlier, CA) on two sites, one infested with M. incognita race 3 isolate Project 77 and the other infested with M. javanica isolate 811. On each site, 1000 F2 seeds were machine planted at 12 m–1 into pre-irrigated beds spaced 0.75 m apart. Beds were formed after application and incorporation of preplant herbicides and 12-12-12 fertilizer at 112 kg ha–1. Plants were furrow irrigated and machine cultivated for optimal growth. Adjacent to test rows, susceptible Henderson Bush was planted to check for uniformity of nematode infestation in the test sites. Root systems of test plants and susceptible control plants were scored for presence and/or absence of primary galling 90 d after planting.

Statistical Analysis
Chi-square goodness-of-fit values for segregation and independence of inheritance of resistance genes were calculated using Statistix 8 (Analytical Software, 2003). Yates correction for continuity was used to determine chi-square values since all ratios involved two classes. Egg and egg mass data were analyzed with SAS ANOVA program (1989–1996, SAS Institute, Inc., Cary, NC).

Nematode egg data were transformed by a log10(x + 1) conversion before analysis to equalize variances among treatment means because of the positive correlation between means and variances. For data with significant treatment F values (p ≤ 0.05), treatment means were compared using least significant difference (LSD) tests at 5%. To examine effects of resistance genes singly and in combination on nematode reproduction, contrasts were performed on log-transformed egg data of the 19-line subset of RILs. Contrast estimates, Q, were calculated using the formula Q = {Sigma}ciYi, where ci is the set of contrast coefficients and Yi is the set of treatment totals. Scheffe's test (Steel and Torrie, 1980) was used to compare |Q| with the critical value for a contrast, SsQ, calculated using the formula SsQ = [ftF{alpha}(ft,fe){Sigma}ci2rs2]–1/2, where ft and fe are treatment and error degrees of freedom, F is the tabulated value for error rate of {alpha}, r is the number of replicates, and s2 is the mean square for error. SsQ was calculated at {alpha} = 0.05 and 0.01 for each contrast. If |Q| was larger than SsQ for a contrast, the null hypothesis was rejected and the contrast was considered significant, either at the 5 or 1% probability level.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Phenotypes and Inheritance of Resistance
A summary of segregation data and chi-square goodness-of-fit values for all populations tested for L-136-derived resistance to M. incognita and M. javanica is presented in Table 1 . Every chi-square test showed no significant difference (p > 0.05) between observed and expected ratios of resistant to susceptible reactions. Five of the 11 tests had probabilities equal to 1.0, with identical observed and expected ratios. Of the remaining six tests, only one had a probability of <0.50. On the basis of these chi-square tests, there was no statistical evidence to reject the models hypothesized for the segregation data.


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  		Table 1. Classification for resistance to Meloidogyne incognita and M. javanica reproduction and root galling of parents and segregating populations derived from crosses between susceptible ‘Henderson Bush’ and resistant ‘L-136’.

 
Meloidogyne incognita Galling
Tests of F1, F2, and RIL populations inoculated with M. incognita showed that a single, dominant gene, designated here as Mig-1, controlled resistance to primary galling in L-136 (Table 1). In greenhouse pot tests, susceptible plants displayed a characteristic swelling or enlargement of most or the entire primary root (Fig. 1A, C). For many susceptible plants, the root swelling was most severe near the root–stem junction, with galled tissue breaking the soil surface. Often, there was swelling of some larger secondary roots, but usually proximal to the primary root. In contrast, the lack of galling observed on the primary root of resistant plants was complete (Fig. 1B), even with plants supporting significant nematode reproduction, as evidenced by the large numbers of egg masses (see Fig. 1D for equivalent reaction with M. javanica). Dominance of this trait was indicated by resistant reactions in the seven pot-tested F1 plants. Although the RIL segregation was skewed (p = 0.099), an expected ratio of 1:1 for a single gene could not be rejected. The F2 segregation in the field test provided strong evidence of a single gene, with p = 0.734 for a 3:1 ratio of nongalled to galled plants (Table 1). Enough F2 plants were tested so that alternative hypotheses of 13:3 and 11:5 could be rejected at 5%. In the field test, susceptible plants in check rows confirmed that the M. incognita population was uniform and intensive enough for a reliable evaluation of F2 segregation. Susceptible, galled roots were easily distinguished from resistant, nongalled roots. There was variability in the severity of galling, and plants with moderate galling were scored as susceptible. The observed ratio was 634 nongalled:205 galled plants (Table 1), a five-plant difference from the expected ratio for a single, dominant gene.

Meloidogyne javanica Galling
A single dominant gene in L-136, designated here as Mjg-1, controlled resistance to primary galling induced by M. javanica in pot and field tests (Fig. 1D). Lack of galling of the primary root in pot-tested F1 plants was similar to that of the resistant parent L-136, providing evidence for dominance of this trait. Segregation of the RIL population supported an expected 1:1 ratio for a single gene (p = 0.708). In the M. javanica field test, galling of roots in check rows was uniform and severe, indicating that the infection level was adequate for testing. In this test, 456 F2 plants segregated 341 nongalled to 115 galled, with a one-plant difference separating observed and expected ratios for one dominant gene (Table 1).

Meloidogyne incognita Reproduction
In the F1, F2, F2:3, and RIL tests, a single recessive gene in L-136, designated here as mir-1, was found to suppress reproduction of M. incognita (Fig. 1B, C). Reproduction on the seven resistant L-136 plants (162,100 mean eggs per plant and 1340 mean eggs per gram root) was less (p = 0.05, based on log10 n + 1 transformed data) than on both the seven F1 (701,100 mean eggs per plant and 6450 mean eggs per gram root) and susceptible parent Henderson Bush (563,700 mean eggs per plant and 5710 mean eggs per gram root) plants. The F1 and Henderson Bush were not statistically separable, indicating this trait was recessive. Chi-square tests of numbers of egg masses on pouch-tested F2 and F2:3 plants supported a single, recessive gene hypothesis (Table 1). Segregation of F2 plants fit an expected 1R:3S ratio (p = 1.000). Among pooled segregating F3 families, the observed ratio of 29 resistant plants to 75 susceptible plants differed from the expected ratio by three plants (p = 0.571). The set of 119 RILs, tested in pots and classified for resistance to reproduction based on presence or absence of egg masses, segregated 57 resistant to 62 susceptible (Table 1), differing from an expected 1:1 ratio for a single gene by three plants (p = 0.708). In the pouch screens, values for mean, range, and standard deviation of resistant and susceptible F2 and F3 plants were comparable to parental values, providing evidence that their classification was accurate. In the F2 test, the mean, range, and standard deviation for egg masses per plant were 22.1, 5 to 55, and 15.1 for 10 L-136 plants; 178.6, 126 to 280, and 45.9 for 10 Henderson Bush plants; 31.9, 2 to 55, and 15.4 for 75 resistant F2 plants; and 149.6, 56 to 402, and 65.7 for 223 susceptible F2 plants. The F3 family screens for egg masses per plant are given in Table 2 , together with the mean egg masses and total eggs per plant and eggs per gram root values for the F2 plants used to derive the F3 families. The F3 family screen revealed that one F2 plant scored as resistant was in fact susceptible and one F2 plant scored as susceptible was resistant, indicating an approximately 8% probability of misclassification in the F2. Misclassifying the susceptible plant resulted from fungal contamination that covered part of the root system and reduced the number of egg masses that could be counted. We found no obvious explanation for the misclassified resistant plant, but there were probably additional resistant plants with egg mass numbers above the highest score of the resistant parent. Precision in classifying F3 plants was increased by also scoring for secondary galling. Twenty out of 327 F3 plants, or 6%, would have been misclassified on the basis of egg mass counts alone.


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  		Table 2. Production of Meloidogyne incognita egg masses and eggs on susceptible ‘Henderson Bush,’ resistant ‘L-136,’ and their F2 and F2:3 families tested in seed growth pouches.

 
The reduction in M. incognita egg production on plants with gene mir-1 ranged from 70 to almost 90% compared with plants without the gene. In the F1 test, mean total eggs per plant on the resistant parent L-136 was 29% of susceptible Henderson Bush and 23% of their F1. Mean egg masses on resistant F2 plants was 21% of susceptible F2 plants, mean total eggs per plant on resistant F2 plants that were transferred to pots was 12% of susceptible F2 plants, and mean egg masses on resistant F3 plants was 13% of susceptible F3 plants (Table 2). These comparisons indicate that effectiveness of gene mir-1 was the same in the presence of genes Mig-1 and Mjg-1, as observed with L-136, and in their absence, as observed on resistant F2 and F3 plants derived from the RILs possessing only gene mir-1.

Meloidogyne javanica Reproduction
Resistant parent L-136 and derived F1 plants inoculated with M. javanica were not statistically different and were more resistant (p = 0.05, based on log10 n + 1 transformed data) than susceptible parent Henderson Bush for both total eggs per plant and eggs per gram of root. Mean total eggs per plant on L-136 (449,600) was 71% of Henderson Bush (635,100), and the F1 mean (427,800) was 67% of Henderson Bush. Because resistance to M. javanica reproduction was much less effective than resistance to M. incognita, we made no attempt to analyze this trait in the same manner, given the difficulties encountered in classifying F2 and F3 plants for M. incognita resistance where a wider separation of phenotypes existed. Resistance to M. javanica reproduction in the RIL population did not vary on the basis of visual evaluation of egg masses, and all lines appeared susceptible. However, a significant range in M. javanica egg production was found in the RIL subset (Table 3 ), in which mean total eggs on the most resistant line was 45% of that on the most susceptible line.


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  		Table 3. Ranking of mean total eggs per plant of Meloidogyne incognita and M. javanica on F2:7–9 recombinant inbred lines from the cross of susceptible ‘Henderson Bush’ x resistant ‘L-136’ representing eight nematode resistance genotypes (see Table 4) identified by their phenotypic classification for resistance.

 
Independence of Genes mir-1, Mig-1, and Mjg-1 in RILs
Phenotypic classification of the 119 RILs for resistance to primary galling and reproduction of M. incognita and resistance to primary galling of M. javanica resulted in placement of the RILs into eight genotypic groups (Table 4 ). Lines within a group were similar in their response to infection and presumed to carry a unique combination of the three resistance genes, mir-1, Mig-1, and Mjg-1.


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  		Table 4. Distribution by genotype of F2:7–9 recombinant inbred lines (RILs) from the cross of susceptible ‘Henderson Bush’ x resistant ‘L-136’ according to their phenotypic classification for resistance to nematode reproduction and galling.

 
Plants in Group 1 (Genotype 1), with 11 lines, suppressed reproduction of M. incognita but displayed galling of the primary root. Although galled, roots of these plants were much cleaner than roots of plants that supported reproduction, due to the reduction in numbers of egg masses with adhering soil particles. Galling of the primary root was often less severe compared with fully susceptible plants like Henderson Bush, and galled tissue did not break down from secondary infection as readily. There was a lack of secondary galling. In response to M. javanica infection, primary root galling was similar to susceptible Henderson Bush. Plants in Group 1 carried mir-1, but not Mig-1 and Mjg-1. A line from this group was used to derive the F2 and F2:3 populations for pouch testing. Plants of the nine lines in genotype Group 2 responded to M. incognita infection similar to Group 1 plants but controlled galling to M. javanica. Plants in Group 2 carried mir-1 and Mjg-1, but not Mig-1. Plants in Groups 3 (15 lines) and 4 (15 lines) were fully susceptible to M. incognita. Primary and secondary galling, together with presence of egg masses and adhering soil particles, were evident on roots. Severely galled primary roots were prone to rotting. Plants in these groups differed in primary root galling response to M. javanica, being present in Group 3 and absent in Group 4. Thus, Group 3 plants had the same genotype as susceptible Henderson Bush (no resistance genes), and Group 4 plants possessed only gene Mjg-1. Plants in Groups 5 (22 lines) and 6 (15 lines) were resistant to M. incognita, carrying mir-1 and Mig-1, but differed in response to M. javanica primary root galling, with Group 5 lacking Mjg-1 (galled) and Group 6 possessing Mjg-1 (nongalled). Group 6 plants had the same genotype as the resistant parent L-136, with the full complement of resistance genes. Plants in Groups 7 (14 lines) and 8 (18 lines) were susceptible to reproduction and resistant to galling by M. incognita. In response to M. javanica, Group 7 plants galled and Group 8 plants were nongalled, indicating that Group 7 plants possessed gene Mig-1, and Group 8 plants possessed genes Mig-1 and Mjg-1.

Chi-square tests provided evidence for complete independence of inheritance of genes mir-1, Mig-1, and Mjg-1 on the basis of phenotypic distribution of the 119 RILs (Table 5 ). In four of the six tests, p > 0.50. The other two tests had lower p values, with ratios skewed from the 1:1 ratio expected for complete independence, but these could not be used to reject the hypothesis that genes mir-1, Mig-1, and Mjg-1 were unlinked.


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  		Table 5. Tests for independence of inheritance of genes mir-1, Mig-1, and Mjg-1 based on phenotypic classification (see Table 4) of F2:7–9 recombinant inbred lines from the cross of susceptible ‘Henderson Bush’ x resistant ‘L-136’.

 
Effects of Genes mir-1, Mig-1, and Mjg-1 on Nematode Reproduction
Effects of the three genes on M. incognita and M. javanica reproduction were examined in detail with a subset of 19 indeterminate RILs consisting of two or three lines each of the eight genotypic groups. A visual assessment for phenotypes was made and roots were processed for egg production. The 19 lines are ranked from highest to lowest for transformed mean total eggs per root system of each nematode species, plus phenotypic reactions and the putative genotype for each line (Table 3).

Meloidogyne incognita Reproduction
Egg production of M. incognita on the most resistant line was 3% of that on the most susceptible line. Egg production on lines with Genotypes 4, 7, and 8 was not different than on Genotype 3 lines, equivalent to susceptible Henderson Bush. None of these genotypes carried mir-1, but Genotypes 7 and 8 carried Mig-1. Significant variability occurred among lines within Genotypes 4, 5, and 6, according to the LSD test. Lines within each of the remaining genotypes did not differ in their effects on M. incognita egg production. The line ranking confirmed the suppressive effect of gene mir-1 on reproduction, with the 9 lines carrying mir-1 (Table 3) more resistant than the 10 lines without mir-1. Mean total egg numbers on the lines with mir-1 was 24% of that on lines without mir-1, similar to the parental values in the F1 test. The LSD test, however, did not separate the two adjacent lines with and without mir-1. In the absence of mir-1, there was no evidence of suppression of M. incognita egg production on lines with Mig-1 (Genotypes 7 and 8), with four of the five lines most susceptible to M. incognita possessing Mig-1. In the presence of mir-1, however, lines with Mig-1 (Genotypes 5 and 6) (Table 3) were 60% more resistant than lines without Mig-1 (Genotypes 1 and 2). The two adjacent lines with and without Mig-1 did not separate in the LSD test. Mjg-1 controlling galling to M. javanica was distributed randomly among the ranked lines and had no measurable effect on M. incognita egg production.

Meloidogyne javanica Reproduction
Egg production of M. javanica on the most resistant line (Genotype 2) was 45% of that on the most susceptible line (Table 3) and differed (p = 0.05) from the two most susceptible lines (Genotypes 4 and 7). Within-genotype variability was only found among lines of Genotype 7. The five lines with Genotypes 2 and 6 were grouped together as most resistant, and these lines possessed both Mjg-1 and mir-1 (Table 3). The remaining 14 lines were all more susceptible and possessed only one or neither of the Mjg-1 and mir-1 genes. The 6 most susceptible of these 14 lines lacked mir-1. Mig-1 controlling M. incognita galling was distributed randomly among the ranked lines and had no measurable effect on M. javanica egg production.

Contrast Analysis
Contrast analysis using Scheffe's method (Steel and Torrie, 1980) was applied as a conservative statistical test for comparing the effects of genes in different combinations across genotypes that may not be apparent from the LSD tests. Coefficients (Q) for the most pertinent contrasts and the corresponding required Scheffe's critical value and levels of significance for the two subsets of log-transformed egg data (Table 3) are presented in Table 6 . For M. incognita egg production, the contrasts of lines with and without Mjg-1 and with and without Mig-1 had low coefficients below the required value for significance, confirming that these genes had no effect on reproduction of M. incognita. However, the contrast of lines with and without mir-1 was highly significant, with a coefficient more than double the required critical value, confirming the strong suppression of M. incognita egg production by this gene. The contrast of lines with both mir-1 and Mig-1 versus all other lines was also highly significant, due mostly to the influence of mir-1. Mig-1 did have a significant suppressive effect when combined with mir-1 and contrasted with lines that carried only mir-1, whereas the contrast of lines with and without Mig-1 in the absence of mir-1 was not significant.


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  		Table 6. Effects of resistance genes mir-1, Mig-1, and Mjg-1 on reproduction of Meloidogyne incognita and M. javanica in F2:7–9 recombinant inbred lines (RILs) from a cross of susceptible ‘Henderson Bush’ x resistant ‘L-136’ determined by contrast analysis and Scheffe's test for significance.{dagger}

 
For M. javanica egg production, a significant contrast was found (Table 6) only between the five lines with both mir-1 and Mjg-1 (Genotypes 2 and 6), which supported 61% of mean eggs per plant, and the five lines without these two genes (Genotypes 3 and 7), comparable to the reduced egg production on resistant L-136 (71%) and the F1 (67%). The contrast between lines with and without mir-1 in the presence of Mjg-1 was not significant, however, and other nonsignificant contrasts indicated that neither of these genes alone suppressed M. javanica egg production (Table 6).


   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Results from this study reveal independent genetic control of resistance to root galling and resistance to reproduction of root-knot nematodes in lima bean. The primary root severe galling response is apparently a systemic response to initial nematode infection not requiring normal nematode reproduction that occurs on susceptible roots. Nematode reproduction occurred independently of this response, indicating that different plant response pathways govern the two infection processes. This lima bean system provides an opportunity to investigate the unique aspects of the differentiated pathways in the infection response. While this relatively uncommon phenomenon was reported previously to occur in common bean, soybean, and cotton, on the basis of phenotype observations, our study provides the first genetic analysis of the trait determinants for these unique resistance phenotypes.

Three independently assorting genes for resistance were identified in the line L-136. Genotypes in the RILs possessing all three genes confirmed that selection for broad-based nematode resistance can be achieved in lima bean by pyramiding the genes, as we demonstrated previously with the development of lima bean cultivar Cariblanco N (Helms et al., 2004). Cariblanco N was developed without knowledge of the genetic determinants of the resistance and required breeding selection over many years. The genetic control of the resistance traits reported here should facilitate efficient breeding for comprehensive nematode resistance in lima bean. In addition, common bean and other molecular markers are being applied to these lima bean populations for trait associations and marker-assisted selection to pyramid the nematode resistance genes efficiently.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
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Received for publication July 13, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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