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a Dep. of Botany and Plant Sciences, Univ. of California, Riverside, CA 92521-0124 USA
b Dep. of Nematology, Univ. of California, Riverside, CA 92521-0415 USA
philip.roberts{at}ucr.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Abbreviations: J2, second stage juveniles of root-knot nematodes • LEGR, log10 (n + 1) of number of nematode eggs per gram of fresh root • LLGR, log10 (n + 1) of number of J2 per gram of fresh root • LTOT, log10 (n + 1) of number of eggs per root system • LSM, least-squares mean
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Single gene resistance may not be durable, and reliance on such a system is risky (Sasser, 1980). Recent identification of several isolates of M. incognita which overcome the resistance conferred by Rk (Roberts et al., 1995) indicates that Rk may become less effective for managing M. incognita in the future. Also, resistance conferred by Rk is only partially effective against California isolates of M. javanica (Roberts et al., 1997). These facts support the need to identify additional sources of resistance to root-knot nematodes in cowpea. Breeding line IT84S-2049 possesses a higher level of resistance to both Rk-avirulent and Rk-virulent isolates of M. incognita and to M. javanica than cultivars having Rk (Roberts et al., 1997). The resistance of IT84S-2049 is conferred by a single dominant gene (Rk2) allelic to, or within 0.17 map units of, the Rk locus (Roberts et al., 1996). Accessions PI 441917, PI 441920, and PI 468104 also possess higher levels of resistance to M. incognita than Rk-cultivar Mississippi Silver (Fery et al., 1994). Genetic analysis of these accessions indicates that this heightened resistance is also conferred by a single dominant allele at the Rk locus. These sources of heightened resistance (IT84S-2049 and the three PI's) are poorly adapted to commercial production in the USA, having low yields, non-marketable seed types, and other undesirable traits which means that a substantial breeding effort will be required to utilize their resistances. These alleles help broaden the genetic base of resistance to root-knot, but additional non-allelic resistance would be desirable to enhance the durability and perhaps the level of nematode resistance in cowpea.
Recently, a new broad-based form of resistance to root-knot nematodes that controls both Rk-virulent and Rk-avirulent isolates of M. incognita and Rk-aggressive isolates of M. javanica was identified in a high yielding, heat tolerant, large seeded, Fusarium wilt resistant "blackeye-type" breeding line H8-8R (Roberts et al., 1997) developed at the University of California, Riverside, by J.D. Ehlers, A.E. Hall, and P.N. Patel. Resistance in UCR H8-8R virtually eliminates galling and reproduction of Rk-avirulent isolates of M. incognita and suppresses galling and reproduction of Rk-virulent isolates of M. incognita and Rk-aggressive isolates of M. javanica by at least half, compared with Rk-type commercial blackeye bean cultivars (Roberts et al., 1992; Ehlers et al., 1996). An understanding of the genetic basis of this resistance will facilitate breeding of cultivars with improved resistance and reveal possibilities for combining resistance traits to obtain more durable or higher levels of resistance. The objective of this study was to determine the genetic basis for the higher level of resistance in UCR H8-8R compared with that observed in cultivars possessing only gene Rk.
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Meloidogyne javanica isolate `Project 811', aggressive toward gene Rk, was collected from a field planted to cowpeas near Chino, San Bernardino County, CA, in the mid-1950s (Thomason and McKinney, 1960). Reproduction by this isolate on Rk-plants is about 50% of its rate on non-Rk plants. This level of reproduction is comparable to other California isolates of M. javanica tested on Rk plants. Isolate Project 811 is considered to be aggressive on Rk plants rather than specifically virulent, a condition it exhibits on several other resistant host plants.
Species identity of the isolates was confirmed by isozyme phenotyping and by a host differential test as described previously (Roberts et al., 1996).
Plant Materials and Crosses
Breeding line UCR H8-8R was developed by the pedigree method from the cross UCR breeding line 336 x UCR breeding line 1393. UCR 336 was obtained from the cross `CB5' x `CB3'; CB5 possesses the Rk allele derived from `Iron' (Mackie, 1946), and CB3 is susceptible (genotype rkrk). Parental lines of UCR 1393 were Prima, accession TVu4552, and `CB77'. The pedigree of CB77 is PI166146/CB5//CB5, and this line exhibits resistance typical of Rk cultivars.
Genetic analysis of the higher level of resistance observed in H8-8R was performed by examining the F1 and later generations from crosses between H8-8R and genotypes with Rk, CB88 and CB46 (Helms et al., 1991a, b). This enabled identification of any additional resistance or modifying factors in the Rk background. Rk-virulent or -aggressive nematode isolates were used to distinguish the heightened resistance (designated H) phenotype from the Rk-conferred phenotype (designated Rk). Because the identification of the high resistance in H8-8R (Roberts et al., 1992; Ehlers et al., 1996) involved use of both Rk-virulent M. incognita Muller and Rk-aggressive M. javanica Project 811, these isolates were used in the current study.
The F1 generation was grown in a greenhouse and allowed to self to obtain F2 seeds. F2-derived F3 families (F2:3) were obtained from the cross CB88 x H8-8R using selfed seed from randomly selected F2 plants. Depending on the experiment, 10 to 24 individuals per F3 family were evaluated.
Resistance Screening
Resistance assays were conducted with either greenhouse-grown potted plants or a modified growth-pouch technique (Omwega et al., 1988). In the growth-pouch technique, cowpea seeds were either germinated in petri dishes and transferred singly to pouches or placed directly into the pouch and germinated. The pouches were transferred to a walk-in controlled-environment chamber maintained at a constant temperature of 26.7 ± 0.6°C and 16 h of light per day. Pouches were watered once or twice per day with reverse osmosis water. After about 10 to 14 d, when an adequate root system had developed, the pouches were inoculated with approximately 1500 nematode second-stage juveniles (J2). The inoculum was prepared by extracting nematode eggs from tomato roots with NaOCl (Hussey and Barker, 1973) and hatching them in tap water at approximately 27°C in an incubator. The J2s were stored at 15°C until sufficient numbers had been collected to inoculate a test. The stored inoculum was warmed to room temperature and air was bubbled into the vessel to revive the nematodes prior to inoculation. Following inoculation, plants were watered once or twice daily as needed with Hoagland's solution (Hoagland and Arnon, 1950) until a response to the fertilizer was observed. After that, pouches were kept moist with water. Approximately 30 d after inoculation, each pouch was infused with 75 mg L-1 erioglaucine (Sigma Chemical Co., St. Louis, MO), an egg-mass-selective dye. The pouches were kept flooded with dye overnight, drained, and the root systems evaluated visually under an illuminated desk magnifier for numbers of egg masses. Evaluations of number of eggs per root system and per gram of fresh root were made by pulling the root system off the pouch paper insert, followed by root maceration and sieve extraction of the eggs with NaOCl (Hussey and Barker, 1973). A growth-pouch-tested plant required for progeny development was removed from the pouch and transferred to a pot containing modified University of California (U.C.) soil mix C (Matkin and Chandler, 1957) and allowed to self in a greenhouse. For the allelism test to determine the presence of Rk in H8-8R, F2 seeds of the cross CB88 x H8-8R were grown in growth pouches and inoculated with the Rk-avirulent M. incognita isolate Project 77.
Greenhouse pot evaluations were conducted in 2-L peat pots filled with fine blow sand. Resistance was determined by directly assessing nematode reproduction on the cowpea plants or by a tomato bioassay of the soil in which the cowpea plants had been grown. Each pot had a single cowpea plant and was inoculated about three weeks after planting with 70000 to 80000 eggs in 15 mL of water. Five milliliters of the inoculum was delivered with a syringe at three equidistantly spaced places in the pot. The 7-cm-long syringe needle had three holes drilled at 1.5-cm intervals to facilitate uniform inoculation with depth. Each pot was fertilized by a surface application of 18 g of a 17-6-10 controlled release fertilizer (Scotts-Sierra Horticultural Products Co., Marysville, OH). The pots were drip irrigated as needed to maintain optimal growth. Air temperatures in the greenhouse were maintained between 34 to 37°C during the day and 17 to 20°C at night. The duration of the pot tests was about 65 d from the time of inoculation, allowing completion of three nematode generations and production of seed.
For all pot tests except the F2:3 progeny test, resistance was determined by assessing both total eggs per plant and the number of eggs per gram of fresh root. Eggs were extracted with NaOCl (Hussey and Barker, 1973) and counted. In the F2:3 test, reproduction was assessed either by a J2 hatch from whole washed root systems of 90-d-old cowpea plants or by growing tomato plants in the soil following the cowpea plant and by a root-galling bioassay. NaOCl egg extraction and J2 hatching from roots have given similar results in comparative tests in our laboratory. Tomato and other sensitive (high galling) plants have been used as indicator plants for estimating relative soil population levels of root-knot nematode (Godfrey, 1934; McSorley and Parrado, 1983). Prot and Netscher (1978) demonstrated the utility of tomato as an indicator plant to bioassay for root knot in situ.
F3 families derived from F2 plants that were classified as having the higher resistance "H" phenotype consisted of 12 plants per family. This was considered a sufficient number since all plants in a family were expected to be homozygous for the recessive allele. F3 families derived from F2 plants that were classified as having the Rk phenotype consisted of 24 plants per family. This number was considered adequate to distinguish segregating families (assuming a 1:3 segregation ratio) from families where all plants in the family would be homozygous for Rk only.
Roots to be assessed for J2 hatch were carefully washed in tap water and placed onto screens in funnels and arranged randomly in a mist chamber (Ayoub, 1980). Samples remained in the mist chamber for 7 d. Collected J2 were counted in representative subsamples and expressed as the number of J2 per root system or per gram of fresh root. Because a significant number of plants were senescing when the experiment was terminated, only those families with all plants in reasonable condition were evaluated using the J2 hatch to avoid plants with root loss. For five F3 families, reproduction was expressed as J2 per gram of root and log10(n + 1) transformed J2 per gram of root (LLGR).
The tomato bioassay was used for those F2:3 families where significant senescence was observed on one or more plants in a family. The mature, 90-d-old cowpea plant was cut off above the soil line and a 4- wk-old seedling of susceptible tomato cv. UC82 was transplanted into the pot. The tomato was drip irrigated and fertilized as described above. After 60 d, the tomato roots were washed in tap water and rated visually for extent of root galling. The 0-to-10 root-galling scale of Bridge and Page (1980) was used but only scores of 1 to 9 were made. Roots rated as 1 had few discrete galls, and roots rated as 9 were heavily galled. The extreme scores were dropped because no plants were found without galling (0 rating) or that had died from severity of infection (10 rating).
A log10 (n + 1) transformation was applied to all egg (not egg mass) and J2 data prior to statistical analyses. This was done in order to equalize variances among treatment means because of the positive correlation between means and variances.
Parental and F1 data were analyzed with the SAS ANOVA program (SAS Institute, Inc., 1985). The F3 family data from pouch experiments and J2 data from the pot test were analyzed with the SAS GLM program because of unbalanced data sets resulting from occasional plant mortality. The SAS GLM procedure generated least-squares means (LSMs) and a table of probability values (p-differential) for the hypothesis that LSM (i) = LSM (j) that allowed placement of families into phenotypic classes for hypothesis testing. Segregation data from the three separate F2:3 tests conducted in growth pouches (Table 1) were pooled after testing for homogeneity of ratio (Gomez and Gomez, 1984).
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Results and discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Evaluation of F1 Generations
In greenhouse pot tests inoculated with M. javanica isolate `Project 811' or M. incognita isolate Muller II, H8-8R had significantly fewer eggs per root system and per gram of root than CB88 or CB46 (Table 3)
. A growth-pouch test with isolate Muller II produced similar results (Table 3). No differences were found among reciprocal F1s, indicating an absence of maternal effects. In all tests, the F1s were not distinguishable from the Rk parents, indicating that the additional resistance in H8-8R is recessive to both M. javanica and M. incognita.
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Random F2:3 consisting of 10 plants per family were evaluated for resistance to Rk-virulent M. incognita isolate Muller II in growth pouches. A classification of the F3 families was possible on the basis of log10(n+1) transformed mean eggs per gram of fresh root (LEGR). The assessment by LEGR gave a more effective separation of parental LSMs and higher F values in the analyses of variance than did egg mass counts. We identified those F3 families that were not significantly different from H8-8R in the test (by a probability of >5%) assuming that these families were homozygous for the recessive allele. Any F3 families significantly different (probabilities of 
5%) from H8-8R were classified as equivalent to CB88, without attempting to distinguish segregating families from families homozygous only for the Rk allele. It would be expected that segregating families would have lower LSMs than families that are homozygous only for the Rk allele, but that they would be significantly greater (probability of 5%) than the LSM of H8-8R. Three separate tests with F2:3 families were consistent with the segregation of a single recessive gene for a higher level of resistance to M. incognita, isolate Muller II. Pooled data are shown in Table 1. When an F3 family LSM was not significantly different from the LSM for H8-8R, it was also significantly lower than the LSM for CB88. Conversely, if the family LSM was significantly higher than H8-8R, it was not significantly different from CB88. One family was not significantly different from either parent, and it was classified as equivalent to H8-8R (this family was significantly different from CB88 at P = 11%).
An F2 population of 100 plants derived from the cross CB88 x H8-8R was assessed for resistance to M. javanica isolate Project 811 in a pot test (Table 4) . One hundred plants in the F2 is more than sufficient to distinguish between two expected ratios: 1:3 for a single recessive gene and 1:15 for duplicate recessive genes—even at P = 0.01 (Little and Hills, 1978). The criterion used to classify plants was to assign a plant to either parental class if the plant score (either LTOT or LEGR) was within one standard deviation of the parental mean. Of the 100 plants tested, only two were considered unclassifiable, falling between the parental classes. These results indicate that the greenhouse pot test was more effective than the pouch test in assessing resistance levels of single plants. As assessed by either LTOT or LEGR, this F2 pot test indicated the additional resistance in H8-8R to M. javanica isolate Project 811 is controlled by a single recessive gene (Table 4).
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The results of the F3 J2 hatch and bioassay tests with M. javanica were combined to assess inheritance (Table 7) . The 20 F3 families segregated 1:2:1, thereby confirming that the additional resistance to M. javanica in H8-8R is controlled by a single recessive gene unlinked to the Rk gene. Further, pooled data from 9 of the 10 segregating F3 families (the segregating family evaluated by J2 hatch was excluded) showed that this resistance fits a single gene model. These data also indicated that although the procedures used to score F2 plants in the M. javanica pot test (Table 4) were an improvement over the F2 test conducted in growth pouches that had relied on egg mass counts, some F2 plants were still misclassified (four of 20 in the greenhouse pot test compared with 14 of 25 in the growth pouch test).
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Pedigree Source of Heightened Resistance in H8-8R
The single recessive gene identified in this study could be viewed as a modifier gene that operates either as a recessive enhancer of gene Rk or as a dominant suppressor of Rk (Roberts et al., 1997). An alternative hypothesis is that this recessive gene confers resistance that is independent of the resistance controlled by the Rk gene, and that the two genes combine to give the high level of resistance observed in H8-8R. To test the second hypothesis, examination of the pedigree suggests that UCR 1393, and not UCR 336, donated the recessive resistance gene. UCR 336 was the result of a cross between a susceptible parent (CB3) and a parent known to carry only the Rk gene (CB5). Among the parental lines used to develop UCR 1393, the most likely donor(s) of a resistance gene unlinked to the Rk gene would be Prima and TVu4552. CB77 would be an unlikely source because it carries Rk but does not have the resistance of H8-8R (data not shown). We therefore hypothesize that the Rk allele most likely came from UCR 336, and the recessive resistance allele from UCR 1393.
A growth-pouch screen of TVu4552 and Prima, inoculated with Rk-avirulent M. incognita isolate Project 77, was conducted to determine the probable source of the recessive allele and the nature of its resistance. In this test, the susceptible check (CB3) had a mean egg mass count of 89 (range 37–152), the nematode was effectively controlled by Rk-resistant CB46 (mean of 1; range 0–3), while Prima was susceptible (mean of 86; range 27–155). TVu4552 had a mean of 35 (range 14–50) egg masses per root system. Analysis of variance of these data showed that TVu4552 was significantly more resistant than CB3 but significantly more susceptible than CB46. Prima was not statistically different from CB3. These results suggest that TVu4552 carries some resistance, possibly the recessive gene, but not Rk.
TVu4552 was crossed reciprocally to susceptible CB3 to test whether the resistance of TVu4552 is recessive and therefore the probable donor of the gene identified in H8-8R. Reciprocal F1s, the parents and a known Rk genotype, CB46, were screened in growth pouches by Rk-avirulent M. incognita Project 77. The results indicated the moderate resistance observed in TVu4552 is recessive (Table 8) . The symbol rk3 is proposed for this recessive resistance gene and the probable genotype of TVu4552 is rkrkrk3rk3 (Table 2).
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If the resistance observed in H8-8R is the result of a combination of gene Rk and an unlinked resistance (rk3) gene, one would expect four genotypic classes among the H-lines: (1) Rk-rk3rk3, (2) Rk-Rk3-, (3) rkrkrk3rk3, and (4) rkrkRk3-. Genotypes 1, 2, and 4 were identifiable in the field screen, producing the H8-8R, Rk, and susceptible phenotypes, respectively. Genotype 3 may not have been observed because lines with that gene combination may not have been developed by chance or because the resistance conferred by the recessive gene on its own was too low against this nematode isolate to be identified in a field screen. In the latter case, the resistance of Genotype 3 plants may not have been distinguishable from Genotype 4 plants at the field level of detection.
This is the first report of the occurrence of root-knot nematode resistance in cowpea not linked to the Rk locus. The broadened resistance conferred by the combination of genes Rk and rk3 is an important finding in light of the emergence of Meloidogyne spp. isolates that have become virulent on the Rk gene. Identification of an independent resistance locus opens up the possibility for new gene combinations that may provide resistance that is more effective than resistance based on Rk. For example, it is possible that combining rk3 with the Rk2 resistance discovered by Roberts et al. (1996), which confers a higher level of resistance than Rk to Rk-virulent M. incognita and M. javanica, could lead to an even higher level of resistance that approaches immunity to virulent–aggressive isolates.
A new cultivar, CB27, has recently been released that has the high level of resistance conferred by Rk and rk3 (Ehlers et al., 2000). SAS Institute 1985
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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Received for publication February 23, 1999.
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResults and discussionREFERENCES |
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