Clustering, haplotype diversity and locations of MIC-3: a unique root-specific defense-related gene family in Upland cotton (Gossypium hirsutum L.)

MIC-3 is a recently identified gene family shown to exhibit increased root-specific expression following nematode infection of cotton plants that are resistant to root-knot nematode. Here, we cloned and sequenced MIC-3 genes from selected diploid and tetraploid cotton species to reveal sequence differences at the molecular level and identify chromosomal locations of MIC-3 genes in Gossypium species. Detailed sequence analysis and phylogenetic clustering of MIC-3 genes indicated the presence of multiple MIC-3 gene members in Gossypium species. Haplotypes of a MIC-3 gene family member were discovered by comparative analysis among consensus sequences across genotypes within an individual clade in the phylogram to overcome the problem of duplicated loci in the tetraploid cotton. Deficiency tests of the SNPs delimited six A_t-genome members of the MIC-3 family clustered to chromosome arm 4sh, and one D_t-genome member to chromosome 19. Clustering was confirmed by long-PCR amplification of the intergenic regions using A_t-genome-specific MIC-3 primer pairs. The clustered distribution may have been favored by selection for responsiveness to evolving disease and/or pest pressures, because large variants of the MIC-3 gene family may have been recovered from small physical areas by recombination. This could give a buffer against selection pressure from a broad range of pest and pathogens in the future. To our knowledge, these are the first results on the evolution of clustering and genome-specific haplotype members of a unique cotton gene family associated with resistant response against a major pathogen.     

Histochemical Localization and Nematoxicity of Terpenoid Aldehydes in Cotton

In healthy cotton, except for random occasional occurrence in cortical cells, terpenoid aldehydes (TA) are localized in the epidermis and, even there, are absent from the tip 2-4 cm of the root. Since constitutive TA do not occur in the endodermis and stele of the root, they cannot be effective agents against the development of the sedentary stage of the root-knot nematode, Meloidogyne incognita. Within 4 days after inoculation with the root-knot nematode, infection-induced TA accumulated in the endodermis and outer stele. These induced TA were thus localized where they could be effective against the sedentary stage of the nematode. Infection-induced TA accumulation was more rapid and occurred in more stele cells in a resistant cotton cultivar than in two susceptible cultivars.TA extracts from cotton were inhibitory to nematode movement. All second-stage larvae exposed to 1,000 ppm TA for 3 h became rigid, made no movement, and appeared dead. Washing these larvae to remove the TA and incubating them for an additional 24 h did not change their appearance. Shorter exposure times or lower TA concentrations allowed some larvae to recover. Exposing larvae to 10 ppm of TA for 24 h had little effect on them. TA extracted from G. arboreum, a cotton that does not methylate TA, were slightly less inhibitory to the root-knot nematode than TA extracted front G. hirsutum which partially methylates TA.     

Nematicides and Nonconventional Soil Amendments in the Management of Root-Knot Nematode on Cotton

Granular and liquid commercial humates, with micronutrients, and a microbial fermentation product were compared in several combinations with nematicides for their effects on cotton lint yield and root-knot nematode suppression. Fumigant nematicides effectively reduced cotton root galling caused by root-knot nematodes, and cotton lint yields increased. Organophosphates and carbamates were not effective. Occasionally, cotton lint yields were increased or maintained with combination treatments o f humates, micronutrients, and a microbial fermentation product, but galling o f cotton roots by root-knot nematodes was usually not reduced by these treatments.     

QTL analysis for transgressive resistance to root-knot nematode in interspecific cotton (Gossypium spp.) progeny derived from susceptible parents

The southern root-knot nematode (RKN, Meloidogyne incognita) is a major soil-inhabiting plant parasite that causes significant yield losses in cotton (Gossypium spp.). Progeny from crosses between cotton genotypes susceptible to RKN produced segregants in subsequent populations which were highly resistant to this parasite. A recombinant inbred line (RIL) population of 138 lines developed from a cross between Upland cotton TM-1 (G. hirsutum L.) and Pima 3-79 (G. barbadense L.), both susceptible to RKN, was used to identify quantitative trait loci (QTLs) determining responses to RKN in greenhouse infection assays with simple sequence repeat (SSR) markers. Compared to both parents, 53.6% and 52.1% of RILs showed less (P<0.05) root-galling index (GI) and had lower (P<0.05) nematode egg production (eggs per gram root, EGR). Highly resistant lines (transgressive segregants) were identified in this RIL population for GI and/or EGR in two greenhouse experiments. QTLs were identified using the single-marker analysis nonparametric mapping Kruskal-Wallis test. Four major QTLs located on chromosomes 3, 4, 11, and 17 were identified to account for 8.0 to 12.3% of the phenotypic variance (R(2)) in root-galling. Two major QTLs accounting for 9.7% and 10.6% of EGR variance were identified on chromosomes 14 and 23 (P<0.005), respectively. In addition, 19 putative QTLs (P<0.05) accounted for 4.5-7.7% of phenotypic variance (R(2)) in GI, and 15 QTLs accounted for 4.2-7.3% of phenotypic variance in EGR. In lines with alleles positive for resistance contributed by both parents in combinations of two to four QTLs, dramatic reductions of >50% in both GI and EGR were observed. The transgressive segregants with epistatic effects derived from susceptible parents indicate that high levels of nematode resistance in cotton may be attained by pyramiding positive alleles using a QTL mapping approach.     

Effect of Mowing Cotton Stalks and Preventing Plant Re-Growth on Post-Harvest Reproduction of Meloidogyne incognita

The southern root-knot nematode (Meloidogyne incognita) is a major parasite of cotton in the U.S., and management tactics for this nematode attempt to minimize population levels. We compared three post-harvest practices for their ability to reduce nematode population levels in the field, thereby reducing initial nematode population for the next year's crop. The three practices tested were: 1) chemical defoliation before harvest plus cutting cotton stalks after harvest, 2) chemical defoliation plus applying a herbicide to kill plants prior to cutting the stalks, and 3) chemical defoliation without cutting stalks. Experiments were conducted in both the greenhouse and in the field. The greenhouse experiments demonstrated that M. incognita reproduction (measured as egg counts and root gall rating indices) was significantly greater when stalks were not cut. Cutting stalks plus applying herbicide to kill cotton roots did not significantly reduce nematode reproduction compared to cutting stalks alone. In field experiments, cutting stalks reduced egg populations and root galling compared to defoliation without stalk cutting. In a greenhouse bioassay which used soil from the field plots, plants grown in soil from the defoliation only treatment had greater root gall ratings and egg counts than in the stalk cutting plus herbicide treatment. Therefore, we conclude that cutting cotton stalks immediately after harvest effectively reduces M. incognita reproduction, and may lead to a lower initial population density of this nematode in the following year.     

Re-evaluation of the inheritance for root-knot nematode resistance in the Upland cotton germplasm line M-120 RNR revealed two epistatic QTLs conferring resistance

Key message

We report a second major QTL for root-knot nematode resistance in the highly resistant Upland cotton line M-120RNR and show epistasis between two resistant QTLs with different mechanisms conferring resistance.

Abstract

In an earlier study, we identified a major QTL on Chromosome 11 associated with resistance to root-knot nematode in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. Herein, we re-evaluated the genetics of the resistance to root-knot nematode in the M-120 RNR × Pima S-6 population by linkage mapping using recently published SSR markers. The QTL analysis detected two regions significantly associated with the resistance phenotype. In addition to the QTL previously identified on Chromosome 11 (qMi-C11), a major QTL was identified on Chromosome 14 (qMi-C14). The resistance locus on qMi-C11 originated from the Clevewilt parent, while the qMi-C14 locus originated from the other resistant parent, Mexico Wild Jack Jones. The qMi-C14 locus had logarithms of odds score of 17 and accounted for 45 % of the total phenotype variation in egg production. It was also associated with galling index, but the percent variation explained was only 6 %, suggesting that the qMi-C11 locus had a much stronger effect on root gall suppression than egg production, while the qMi-C14 locus had a stronger effect on egg production than galling. The results also suggest that the transgressive segregation observed in the development of Auburn 623 RNR was due to the pyramiding of at least two main effect QTLs as well as an additive-by-additive epistatic effects between the two resistant loci. The SSRs markers tightly linked to the qMi-C11 and qMi-C14 loci will greatly facilitate the improvement of RKN resistance in cotton via marker-assisted breeding.     

Fine mapping QMi-C11 a major QTL controlling root-knot nematodes resistance in Upland cotton

The identification and utilization of a high-level of host plant resistance is the most effective and economical approach to control root-knot nematode (Meloidogyne incognita). In an earlier study, we identified a major quantitative trait locus (QTL) for resistance to root-knot nematode in the M-120 RNR Upland cotton line (Gossypium hirsutum L.) of the Auburn 623 RNR source. The QTL is located in a 12.9-cM interval flanked by the two SSR markers CIR069 and CIR316 on the distal segment of chromosome 11. To construct a fine map around the target region, a bulked segregation analysis was performed using two DNA pools consisting of five individuals, with each being homozygous for the two parental alleles. From a survey of 1,152 AFLP primer combinations, 9 AFLP markers closely linked to the target region were identified. By screening an additional 1,221 F₂ individuals developed from the initial mapping population, the Mi-C11 locus was delimited to a 3.6-cM interval flanked by the SSR marker CIR069 and the AFLP marker E14M27-375. These results further elucidate the genetic fine structure of the Mi-C11 locus and provide the basis for map-based isolation of the nematode resistance gene in M-120 RNR.     

Evaluation of NemX,a New Cultivar of Cotton with High Resistance to Meloidogyne incognita

The level of resistance to root-knot nematode, Meloidogyne incognita, in NemX, a new cultivar of the Acala-type upland cotton, was evaluated in relation to four resistant breeding lines (N6072, N8577, N901, and N903) and four susceptible cultivars (Maxxa, SJ2, Royale, and Prema). In growth pouch tests, an average of only 4 nematode egg masses was produced on roots of NemX or the resistant lines, compared to a significantly higher average of 21 on the susceptible cultivars. In pot tests, the nematode reproduction factor (RF = Pf/Pi) in NemX and the resistant lines averaged 0.7, compared to a significantly higher average of 10 on the susceptible cultivars. Root galling in NemX or other resistant cotton averaged 15%, compared to 74% on the susceptible cultivars, in either pot or field tests. In plots with low levels of nematode infestation (Pi ≤ 150 second-stage juveniles [J2]/500 g soil), lint yield of NemX averaged 1,370 kg/ha and was less than the yield of susceptible Maxxa (1,450 k g /h a ). However, in plots with medium or high levels of nematode infestation (Pi = 151-300 or >300 J2/500 g soil, respectively), yields of NemX decreased only slightly and averaged 1,300 or 1,050 kg/ha, respectively, whereas yields of Maxxa were severely reduced to 590 or 503 kg/ha, respectively. Fusarium wih symptoms were observed on both NemX and Maxxa, and percent occurrence increased with increasing preplant nematode density. In plots with the highest nematode densities, 22% of NemX and 65% of Maxxa plants were wilted. NemX was highly effective against five M. incognita isolates and moderately effective against a sixth isolate that had been exposed to resistant cotton over several seasons. These results showed that NemX is as resistant to M. incognita as the four breeding lines, and much more resistant than the tested susceptible cultivars of cotton.     

Molecular Characterisation and Diagnosis of Root-Knot Nematodes (Meloidogyne spp.) from Turfgrasses in North Carolina,USA

Root-knot nematodes (Meloidogyne spp.) are the most common and destructive plant-parasitic nematode group worldwide and adversely influence both crop quality and yield. In this study, a total of 51 root-knot nematode populations from turfgrasses were tested, of which 44 were from North Carolina, 6 from South Carolina and 1 from Virginia. Molecular characterisation was performed on these samples by DNA sequencing on the ribosomal DNA 18S, ITS and 28S D2/D3. Species-specific primers were developed to identify turfgrass root-knot nematode through simplex or duplex PCR. Four species were identified, including M. marylandi Jepson & Golden in Jepson, 1987, M. graminis (Sledge & Golden, 1964) Whitehead, 1968, M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. naasi Franklin, 1965 through a combined analysis of DNA sequencing and PCR by species-specific primers. M. marylandi has been reported from North Carolina and South Carolina for the first time. Molecular diagnosis using PCR by species-specific primers provides a rapid and cheap species identification approach for turfgrass root-knot nematodes.     

Effects of Site-specific Application of Aldicarb on Cotton in a Meloidogyne incognita-infested Field

Cotton farmers in Missouri commonly apply a single rate of aldicarb throughout the field at planting to protect their crop from Meloidogyne incognita, even though these nematodes are spatially aggregated. Our purpose was to determine the effect of site-specific application of aldicarb on cotton production in a field infested with these nematodes in 1997 and 1998. Cotton yields were collected from sites not treated with aldicarb (control), sites receiving aldicarb at the standard recommended rate of 0.58 kg a.i./ha, and sites receiving specific aldicarb rates based on the soil population densities of second-stage infective juveniles of root-knot nematode. Yields for the standard rate and site-specific rate treatments were similar and greater (P ≤ 0.05) than the control treatment. Less aldicarb was used for the site-specific than the uniform-rate treatment each year—46% less in 1997 and 61% less in 1998. Costs associated with the site-specific treatment were very high compared with the uniform-rate treatment due to a greater number of soil samples analyzed for nematodes. Site-specific application of aldicarb for root-knot nematode management in cotton may pose fewer environmental risks than the uniform-rate application of aldicarb.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

湖南省平江县水稻根结线虫病发生及危害评价

【目的】进一步明确湖南省平江县水稻根结线虫病发生危害情况、病原线虫种类及影响水稻发病的因素。【方法】2020年4—6月,采用5点取样法对湖南省平江县24个乡镇400块田块的水稻根结线虫病发生危害情况进行再调查及病原种类鉴定,并对水稻根结线虫病的病情指数与水稻的栽培方式、土壤类型之间的关系进行了分析。【结果】平江县水稻根结线虫病的发生较为普遍,发现病害的乡镇已由2018年的5个增加到2020年的15个,其中15个乡镇的病田率为10%～100%,病株率为2%～55%,根结指数为0.4～15.4。分离获得的病原线虫种类均为拟禾本科根结线虫。调查还发现,发病水稻在砂壤、中壤、重壤稻田的根结指数明显高于在黏土和轻壤稻田的根结指数,直播、抛秧栽培方式下发病水稻的根结指数明显高于移栽方式下的根结指数。【结论】平江县水稻根结线虫病呈蔓延趋势。本研究为有效防控平江县水稻根结线虫病提供了依据。