Effect of Temperature on the Embryogenesis of Geographic Populations of Rotylenchulus reniformis

The effect of temperature on the embryonic development of three populations of reniform nematode (Rotylenchulus reniformis) from the southeastern United States was studied. The development of eggs from single-cell stage to eclosion of second-stage juvenile was monitored at 20, 25, 30, and 35°C. All populations completed embryogenesis in 7 days at 25°C. The greatest differences among populations in time to completion of embryogenesis were observed at 20 and 35°C. Results at the intermediate temperatures (25 and 30°C) were similar for the three populations. The optimal temperature for embryogenesis was calculated to be 31.4°C for the population from Alabama, 28.4°C for the one from Mississippi, and 37.5°C for the one from South Carolina.     

Suppression of Rotylenchulus reniformis on Cotton by the Nematophagous Fungus ARF

The reniform nematode, Rotylenchulus reniformis Linford &Oliveira, has become a serious threat to cotton (Gossypium hirsutum L.) production in the United States during the past decade. The objective of this study is to isolate fungi from eggs of R. reniformis and select potential biological control agents for R. reniformis on cotton. Soil samples were collected from cotton fields located in Jefferson County, Arkansas. Eight genera of fungi were included in the 128 fungal isolates obtained, and among them were five strains of the nematophagous fungus ARF. The mtDNA RFLP pattern, colony growth characteristics, and pathogenicity indicate the five ARF isolates represent one described strain and one new strain. Light and electron microscopic observations suggest ARF is an active parasite of R. reniformis, with parasitism ranging from 48% to 79% in in vitro tests. Three greenhouse experiments demonstrated ARF successfully suppressed the number of reniform nematodes during the first and second generation of the nematode. Reductions in numbers of R. reniformis on the roots for the seven application rates of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% ARF were 87%, 92%, 94%, 96%, 97%, 98%, and and 98%, respectively.     

Protease inhibitors and reproduction of reniform nematode in pineapple

Endogenous protease inhibitors (PIs) in the roots of Smooth Cayenne pineapple clones may affect the growth of the plant-parasitic nematode Rotylenchulus reniformis . In pineapple, reniform population densities remain atypically near preplant levels for 6–9 months after pineapple planting. A potted plant experiment was conducted to determine if the PI present in pineapple roots affected nematode reproduction and possibly account for the observed nematode field population dynamics. Pineapple PI activity increased for the first 6 months after planting and was higher in nematode-inoculated plants. Nematode reproduction, however, was not correlated to PI activity. In a second experiment, PI activity was concentrated in the portion of the roots nearest the pineapple butt where nematode population densities were highest. The behaviour of the PI in pineapple roots suggested a defensive role, such as systemic acquired resistance, against R. reniformis .     

Molecular and Morphological Characterization and Biological Control Capabilities of a Pasteuria ssp. Parasitizing Rotylenchulus reniformis, the Reniform Nematode

Rotylenchulus reniformis is one of 10 described species of reniform nematodes and is considered the most economically significant pest within the genus, parasitizing a variety of important agricultural crops. Rotylenchulus reniformis collected from cotton fields in the Southeastern US were observed to have the nematode parasitic bacterium Pasteuria attached to their cuticles. Challenge with a Pasteuria-specific monoclonal antibody in live immuno-fluorescent assay (IFA) confirmed the discovery of Pasteuria infecting R. reniformis. Scanning and transmission electron microscopy were employed to observe endospore ultrastructure and sporogenesis within the host. Pasteuria were observed to infect and complete their life-cycle in juvenile, male and female R. reniformis. Molecular analysis using Pasteuria species-specific and degenerate primers for 16s rRNA and spoII, and subsequent phylogenetic assessment, placed the Pasteuria associated with R. reniformis in a distinct clade within established assemblages for the Pasteuria infecting phytopathogenic nematodes. A global phylogenetic assessment of Pasteuria 16s rDNA using the Neighbor-Joining method resulted in a clear branch with 100% boot-strap support that effectively partitioned the Pasteuria infecting phytopathogenic nematodes from the Pasteuria associated with bacterivorous nematodes. Phylogenetic analysis of the R. reniformis Pasteuria and Pasteuria spp. parasitizing a number of economically important plant parasitic nematodes revealed that Pasteuria with different host specificities are closely related and likely constitute biotypes of the same species. This suggests host preference, and thus effective differentiation and classification are most likely predicated by an influential virulence determinant(s) that has yet to be elucidated. Pasteuria Pr3 endospores produced by in vitro fermentation demonstrated efficacy as a commercial bionematicide to control R. reniformis on cotton in pot tests, when applied as a seed treatment and in a granular formulation. Population control was comparable to a seed-applied nematicide/insecticide (thiodicarb/imidacloprid) at a seed coating application rate of 1.0 x 10(8) spores/seed.     

Additional Fertilizer and Nematicide Combinations on Upland Cotton to Manage Rotylenchulus Reniformis and Meloidogyne Incognita in Alabama

Plant parasitic nematodes are major pests on upland cotton worldwide and in the United States. The reniform nematode, Rotylenchulus reniformis and the southern root-knot nematode Meloidogyne incognita are some of the most damaging nematodes on cotton in the United States. Current management strategies focus on reducing nematode populations with nematicides. The objective of this research was to integrate additional fertilizer and nematicide combinations into current practices to establish economical nematode management strategies while promoting cotton yield and profit. Microplot and field trials were run to evaluate fertilizer and nematicide combinations applied at the pinhead square (PHS) and first bloom (FB) plant growth stages to reduce nematode population density and promote plant growth and yield. Cost efficiency was evaluated based on profit from lint yields and chemical input costs. Data combined from 2019 and 2020 suggested a nematicide seed treatment (ST) ST + (NH₄)₂SO₄ + Vydate^® C-LV + Max-In^® Sulfur was the most effective in increasing seed cotton yields in the R. reniformis microplot trials. In R. reniformis field trials, a nematicide ST + (NH₄)₂SO₄ + Vydate^® C-LV at PHS supported the largest lint yield and profit per hectare at $1176. In M. incognita field trials, a nematicide ST + 28-0-0-5 + Vydate^® C-LV + Max-In^® Sulfur at PHS and FB supported the largest lint yields and profit per hectare at $784. These results suggest that combinations utilizing fertilizers and nematicides applied together across the season in addition to current fertility management show potential to promote yield and profit in R. reniformis and M. incognita infested cotton fields.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

不同地区斑点叉尾鮰种内遗传多样性分析

采用RAPD方法对来源于美国三个地区的30条斑点叉尾鮰进行遗传多样性的分析。对60个随机引物进行筛选,选出能够区分地区内以及地区间种群遗传多态性的引物。通过统计学的方法计算分析得出三个地区各自种群内遗传相似率,德克萨斯州为82.52%,阿纳巴马州为80.46%,密西西比州为83.71%。对地区之间种群的遗传分化情况进行t检测分析得出三个地区之间斑点叉尾鮰种群的遗传特性各不相同,其中差异最大的是阿纳巴马州和密西西比州,差异最小的是德克萨斯州和密西西比州。     

Interrelationships of Rotylenchulus reniformis with Rhizoctonia solani on Cotton

The interrelationships between reniform nematode (Rotylenchulus reniformis) and the cotton (Gossypium hirsutum) seedling blight fungus (Rhizoctonia solani) were studied using three isolates of R. solani, two populations of R. reniformis at multiple inoculum levels, and the cotton cultivars Dehapine 90 (DP 90) and Dehapine 41 (DP 41). Colonization of cotton hypocotyl tissue by R. solani resulted in increases (P ≤ 0.05) in nematode population densities in soil and in eggs recovered from the root systems in both 40- and 90-day-duration experiments. Increases in soil population densities resulted mainly from increases in juveniles. Enhanced reproduction of R. reniformis in the presence of R. solani was consistent across isolates (1, 2, and 3) of R. solani and populations (1 and 2) and inoculum levels (0.5, 2, 4, and 8 individuals/g of soil) of R. reniformis, regardless of cotton cultivar (DP 90 or DP 41). Severity of seedling blight was not influenced by the nematode. Rhizoctonia solani caused reductions (P ≤ 0.05) in cotton growth in 40- and 90-day periods. Rotylenchulus reniformis reduced cotton growth at 90 days. The relationship between nematode inoculum levels and plant growth reductions was linear. At 90 days, the combined effects of these pathogens were antagonistic to plant growth.     

Postinfection Development of Rotylenchulus reniformis on Resistant Gossypium barbadense Accessions

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

Host Suitability of Soybean Cultivars and Breeding Lines to Reniform Nematode in Tests Conducted in 2001

Reproduction of reniform nematode Rotylenchulus reniformis on 139 soybean lines was evaluated in a greenhouse in the summer of 2001. Cultivars and lines (119 total) were new in the Arkansas and Mississippi Soybean Testing Programs, and an additional 20 were submitted by C. Overstreet, Louisiana State Extension Nematologist. A second test of 32 breeding lines and 2 cultivars from the Clemson University soybean breeding program was performed at the same time under the same conditions. Controls were the resistant cultivars Forrest and Hartwig, susceptible Braxton, and fallow infested soil. Five treatment replications were planted in sandy loam soil infested with 1,744 eggs and vermiform reniform nematodes, grown for 10 weeks in 10 cm-diam.- pots. Total reniform nematodes extracted from soil and roots was determined, and a reproductive factor (final population (Pf)/ initial inoculum level (Pi)) was calculated for each genotype. Reproduction on each genotype was compared to the reproduction on the resistant cultivar Forrest (RF), and the log ratio [log₁₀(RF + 1) is reported. Cultivars with reproduction not significantly different from Forrest (log ratio) were not suitable hosts, whereas those with greater reproductive indices were considered suitable hosts. These data will be useful in the selection of soybean cultivars to use in rotation with cotton or other susceptible crops to help control the reniform nematode and to select useful breeding lines as parent material for future development of reniform nematode resistant cultivars and lines.     

Genome-Wide Analysis of Simple Sequence Repeats and Efficient Development of Polymorphic SSR Markers Based on Whole Genome Re-Sequencing of Multiple Isolates of the Wheat Stripe Rust Fungus

The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs) are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.     

Genetic variation of Hoplolaimus columbus populations in the United States using PCR-RFLP analysis of nuclear rDNA ITS regions

Hoplolaimus columbus is an important nematode pest which causes economic loss of crops including corn, cotton, and soybean in the Southeastern United States. DNA sequences of the ITS1-5.8S-ITS2 region of ribosomal DNA from H. columbus were aligned and analyzed to characterize intraspecific genetic variation between eleven populations collected from Georgia, Louisiana, North Carolina, and South Carolina. In comparative sequence analysis with clones from either one or two individuals obtained from the eleven populations, we found variability existed among clones from an individual and that clonal diversity observed from within individuals was verified by PCR-RFLP. PCR-RFLP analysis with Rsa I and Msp I restriction enzymes yielded several fragments on 3.0% agarose gel that corresponded to different haplotypes in all populations and the sum of digested products exceeded the length of undigested PCR products, which revealed that ITS heterogeneity existed in a genome of H. columbus. This indicates that heterogeneity may play a role in the evolution of this parthenogenetic species.     

Tolerance to reniform nematode (Rotylenchulus reniformis) race A in pigeonpea (Cajanus cajan) genotypes

The reniform nematode (Rotylenchulus reniformis) is an important pathogen of pigeonpea (Cajanus cajan). Forty‐six medium maturity (mature in 151–200 days at Patancheru, India) pigeonpea genotypes were evaluated for resistance and tolerance to the reniform nematode in greenhouse and field tests, over the period 1990–97. Each genotype was screened for number of nematode egg masses on a 1 (no egg mass = highly resistant) to 9 (> 50 egg masses = highly susceptible) scale. Plant biomass production in carbofurantreated plots was compared with that in non‐treated plots in a field naturally infested with R. reniformis. Pigeonpea genotypes C 11, ICPL 87119 and ICPL 270 were used as nematode susceptible checks. Genotypes with good plant growth, both in nematode‐free and nematode‐infested plots, were identified as tolerant and evaluated for plant growth and yield for at least three years. All the tested genotypes were susceptible (7 and 9 egg mass score). Single‐plant‐selections, based on plant vigour and yield, were made from genotypes showing tolerance to nematode infection. The level of tolerance was enhanced by plant‐to‐progeny row selection for plant vigour and seed yield in a nematode‐sick field for at least three years. The most promising nematode tolerant genotypes produced significantly greater yield and biomass than the locally grown pigeonpea cultivars in fields naturally infested with R. reniformis at two locations. Pigeonpea landraces are considered to be the most likely sources of tolerance to the nematode. These reniform nematode tolerant lines represent new germplasm and they are available in the genebank of pigeonpea at ICRISAT bearing accession numbers ICP 16329, ICP 16330, ICP 16331, ICP 16332, and ICP 16333.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Detection of Suppressiveness against Rotylenchulus reniformis in Soil from Cotton (Gossypium hirsutum) Fields in Texas and Louisiana

Rotylenchulus reniformis is a major problem confronting cotton production in the central part of the cotton belt of the United States of America. In this study, the hypothesis that natural antagonists in some cases are responsible for unusually low densities of the nematode in certain fields was tested by assaying soils from 22 selected fields for the presence of transferable agents in pots containing cotton plants. In one field, soil from four different depth ranges was tested. In the first of two types of assays, 1 part nematode infested soil was added to 9 parts test soil that was left untreated or autoclaved before mixing; this mixture was used to fill pots. In the second type of assay, 1 part test soil was added to 9 or 19 parts pasteurized fine sand, and nematodes were introduced in aqueous suspension. In three experiments representing both types of assay, transferable or autoclavable agent(s) from four fields in South Texas suppressed nematode populations by 48, 78, 90 and 95%. In one experiment, transferable agents in five fields in Louisiana suppressed populations from 37 to 66%. Identification and evaluation of these agents for biological control of R. reniformis merits further study.     

陆地棉优质纤维渐渗系中外源遗传组分的鉴定与分析

鲁原343是一个渐渗了海岛棉优质纤维基因的陆地棉种质,对其渐渗的优质纤维片段进行鉴定,对利用优质纤维渐渗系改良陆地棉品种的纤维品质具有重要意义。本研究以综合性状优良的转基因抗虫棉鲁棉研22号与鲁原343杂交构建作图群体,利用317对SSR引物对鲁原343和鲁棉研22号进行多态性分析,有24对引物表现多态。利用这些引物进一步和TM-1、优质纤维渐渗片段的供体Ash imoun i作比较,初步鉴定出10个SSR位点与海岛棉渐渗有关。利用这些标记分析(鲁棉研22×鲁原343)F2群体的标记基因型和纤维品质性状的关系,6个标记与纤维品质显著相关,涉及到4条染色体。其中与伸长率相关的标记BNL2986(R2=5.87%)和与长度、细度相关的标记NAU751(R2=6.62%,6.01%)同位于16号染色体的连锁群LG1上,标记间距离为17.7 cM;与纤维成熟度相关的标记BNL3590(R2=8.62%)和与成熟度、伸长率相关的标记BNL3971(R2=15.0%,9.79%)位于2号染色体的连锁群LG3上,标记间距离为4.5 cM;与纤维强度相关的标记BNL3279(R2=8.12%)和与细度相关的标记BNL827(R2=13.94%)分别位于LGD02和25号染色体上。     

Suppression of Rotylenchulus reniformis 122-cm Deep Endorses Resistance Introgression in Gossypium

Nine sources of resistance to Rotylenchulus reniformis in Gossypium (cotton) were tested by measuring population density (Pf) and root-length density 0 to 122 cm deep. A Pf in the plow layer less than the autumn sample treatment threshold used by consultants was considered the minimum criterion for acceptable resistance, regardless of population density at planting (Pi). Other criteria were ample roots and a Pf lower than on the susceptible control, as in pot studies. In a Texas field in 2001 and 2002, no resistant accessions had Pf less than the control but all did in microplots into which nematodes from Louisiana were introduced. An environmental chamber experiment ruled out nematode genetic variance and implicated unknown soil factors. Pf in field experiments in Louisiana, Mississippi, and Alabama were below threshold for zero, six and four of the accessions and above threshold in the control. Gossypium arboreum A2–87 and G. barbadense GB-713 were the most resistant accessions. Results indicate that cultivars developed from these sources will suppress R. reniformis populations but less than in pots in a single season.     

Development of microsatellite markers for identifying Brazilian Coffea arabica varieties

Microsatellite markers, also known as SSRs (Simple Sequence Repeats), have proved to be excellent tools for identifying variety and determining genetic relationships. A set of 127 SSR markers was used to analyze genetic similarity in twenty five Coffea arabica varieties. These were composed of nineteen commercially important Brazilians and six interspecific hybrids of Coffea arabica, Coffea canephora and Coffealiberica. The set used comprised 52 newly developed SSR markers derived from microsatellite enriched libraries, 56 designed on the basis of coffee SSR sequences available from public databases, 6 already published, and 13 universal chloroplast microsatellite markers. Only 22 were polymorphic, these detecting 2-7 alleles per marker, an average of 2.5. Based on the banding patterns generated by polymorphic SSR loci, the set of twenty-five coffee varieties were clustered into two main groups, one composed of only Brazilian varieties, and the other of interspecific hybrids, with a few Brazilians. Color mutants could not be separated. Clustering was in accordance with material genealogy thereby revealing high similarity.     

Genetic variation in eastern North American and putatively introduced populations of Ceratocystis fimbriata f. platani

The plant pathogenic fungus Ceratocystis fimbriata f. platani attacks Platanus species (London plane, oriental plane and American sycamore) and has killed tens of thousands of plantation trees and street trees in the eastern United States, southern Europe and Modesto, California. Nuclear and mitochondrial DNA fingerprints and alleles of eight polymorphic microsatellite markers of isolates of C. fimbriata from these regions delineated major differences in gene diversities. The 33 isolates from the eastern United States had a moderate degree of gene diversity, and unique genotypes were found at each of seven collection sites. Fingerprints of 27 isolates from 21 collection sites in southern Europe were identical with each other; microsatellite markers were monomorphic within the European population, except that three isolates differed at one locus each, due perhaps to recent mutations. The genetic variability of C. fimbriata f. platani in the eastern United States suggests that the fungus is indigenous to this region. The genetic homogeneity of the fungus in Europe suggests that this population has gone through a recent genetic bottleneck, perhaps from the introduction of a single genotype. This supports the hypothesis that the pathogen was introduced to Europe through Naples, Italy during World War II on infected crating material from the eastern United States. The Californian population may also have resulted from introduction of one or a few related genotypes because it, too, had a single nuclear and mitochondrial genotype and limited variation in microsatellite alleles.     

Application of Mitochondrial DNA Polymorphism to Meloidogyne Molecular Population Biology

Recent advances in molecular biology have enabled the genotyping of individual nematodes, facilitating the analysis of genetic variability within and among plant-pathogenic nematode isolates. This review first describes representative examples of how RFLP, RAPD, AFLP, and DNA sequence analysis have been employed to describe populations of several phytonematodes, including the pinewood, burrowing, root-knot, and cyst nematodes. The second portion of this paper evaluates the utility of a size-variable mitochondrial DNA locus to examine the genetic structure of Meloidogyne isolates using two alternate methodologies, variable number tandem repeat (VNTR) and repeat associated poiymorphism (RAP) analysis. VNTR analysis has revealed genetic variation among individual nematodes, whereas RAP may provide useful markers for species and population differentiation.