Effects of Ph Gene-Associated versus Induced Resistance to Tobacco Cyst Nematode in Flue-Cured Tobacco

Effects of the systemic acquired resistance (SAR)-inducing compound acibenzolar-S-methyl (ASM) and the plant-growth promoting rhizobacterial mixture Bacillus subtilis A13 and B. amyloliquefaciens IN937a (GB99+GB122) were assessed on the reproduction of a tobacco cyst nematode (TCN- Globodera tabacum solanacearum) under greenhouse conditions. Two sets of two independent experiments were conducted, each involving soil or root sampling. Soil sample experiments included flue-cured tobacco cultivars with (Ph(p)+: NC71 and NC102) and without (Ph(p)-: K326 and K346) a gene (Ph(p)) suppressing TCN parasitism. Root sample experiments examined TCN root parasitism of NC71 and K326. Cultivars possessing the Ph(p) gene (Ph(p)+) were compared with Ph(p)- cultivars to assess the effects of resistance mediated via Ph(p) gene vs. induced resistance to TCN. GB99+GB122 consistently reduced nematode reproductive ratio on both Ph(p)+ and Ph(p)- cultivars, but similar effects of ASM across Ph(p)- cultivars were less consistent. In addition, ASM application resulted in leaf yellowing and reduced root weight. GB99+GB122 consistently reduced nematode development in roots of both Ph(p)+ and Ph(p)- cultivars, while similar effects of ASM were frequently less consistent. The results of this study indicate that GB99+GB122 consistently reduced TCN reproduction in all flue-cured tobacco cultivars tested, while the effects of ASM were only consistent in Ph(p)+ cultivars. Under most circumstances, GB99+GB122 suppressed nematode reproduction more consistently than ASM compared to the untreated control.     

Cross-resistance in Cell Lines of Nicotiana sylvestris Selected for Resistance to Individual Antibiotics

Cell lines initially selected for resistance to the antibioticskanamycin, streptomycin and chloramphenicol, were each testedfor resistance to several different antibiotics. Only the kanamycinresistant lines showed any cross-resistance to other antibiotics.The three lines tested were resistant to streptomycin and neomycin,while one of them, KR103, was also resistant to chloramphenicolearly in its history, although this resistance was subsequentlylost. None of the lines showed any resistance to cycloheximide. Nicotiana sylvestris, cell culture, cross-resistance, antibiotics, chloramphenicol, kanamycin, streptomycin, neomycin, cycloheximide, cytoplasmic mutants, callus     

大豆胞囊线虫抗性基因定位与克隆研究进展

大豆胞囊线虫（soybean cyst nematode,SCN）是大豆生产上一种危害严重的世界性害虫,能给大豆生产造成极大损失。大豆抗性品种选育是防治其措施中最经济、有效的方法。大豆SCN抗性的分子遗传学研究是开展大豆SCN抗性分子育种的理论基础,本文针对SCN抗性基因定位和克隆两个方面的研究现状进行了综述,并对当前研究中存在的问题及发展前景进行了讨论与展望。     

Development of Selected Tobacco Cyst Nematode Isolates on Resistant and Susceptible Cultivars of Flue-cured Tobacco

Tobacco cyst nematode (Globodera tabacum solanacearum) isolates were collected from 11 locations in Virginia, 3 in North Carolina, and 1 in Maryland. Isolates from each location were maintained and increased on flue-cured tobacco, Nicotiana tabacum cv K326. Plants of flue-cured tobacco cultivars K326 (susceptible) and NC567 (resistant) were each inoculated with 6,000 G. t. solanacearum eggs/plant. Tests were conducted over one (6 weeks) or two (14 weeks) generations of the nematode. Shoot and root weights and the number of nematodes present within a 1-g subsample of feeder roots were recorded at completion of the tests. Nematode counts were categorized by nematode life stage (vermiform, swollen, pyriform, and adult). Although significant differences in nematode development were detected among isolates, differences were not consistent across experiments. Results indicate similar virulence among G. t. solanacearum isolates on resistant and susceptible flue-cured tobacco cultivars. Therefore, plant breeders may effectively use a single G. t. solanacearum isolate when screening tobacco germplasm for resistance.     

Determination of Resistance or Susceptibility of Soybean Genotypes to Soybean Cyst Nematode, Heterodera glycines, Race 3

Resistance and/or susceptibility of several soybean genotypes to soybean cyst nematode (SCN), Heterodera glycines, race 3 was investigated. Three, 11, and 4 soybean genotypes were identified as resistant, moderately resistant, and susceptible, respectively. Resistant plants had fewer cysts on the roots and more root nodules, with the converse in susceptible plants. Reduction in yield of six extensively grown soybean cvs in Alabama was from 10 to 56% compared with the north Alabama average; resistant cv. ‘Bedford’ showed a yield loss of 10%. Cyst numbers were negatively correlated with nodule numbers and other agronomic characters. Nodules and other yield components were positively correlated except for non-correlation of nodules and number of pods with plant height in the 1980 experiment.     

利用KASP标记筛选含rhg1和Rhg4位点的大豆抗病资源

大豆胞囊线虫(SCN,soybean cyst nematode)病是一种世界性大豆病害,培育抗SCN大豆品种是防治SCN的重要措施.本研究利用来自抗SCN主效位点rhg1和Rhg4的2个KASP标记,对487份大豆材料进行筛选,选择含有抗性位点且农艺性状优异的材料;通过室内接种大豆胞囊线虫2号、4号、5号生理小种和新...     

Nonhost Root Penetration by Soybean Cyst Nematode

A total of 66 plants in 50 species were inoculated with eggs and juveniles of soybean cyst nematode, Heterodera glycines. Roots were stained and observed for penetration and development of the nematode. Twenty-six plants were not penetrated; twenty-three were penetrated, but there was no development of the nematode; eight were penetrated with some nematode development; two were penetrated and had considerable nematode development, but few nematodes, if any, matured; and seven were penetrated with many nematodes maturing. The penetration of nonhosts may imply some susceptibility and that populations eventually would build up on the penetrated plants. Plants not penetrated may be useful as rotation plants because no reproduction would occur.     

Amplification of repetitive DNA from Nicotiana plumbaginifolia in asymmetric somatic hybrids between Nicotiana sylvestris and Nicotiana plumbaginifolia

Summary Asymmetric somatic hybrids were obtained between a chlorophyll-deficient mutant of Nicotiana sylvestris (V42) and a nitrate-reductase (NR)-deficient line of N. plumbaginifolia (cnx20 or Nia26), using each of the parents alternately as the irradiated donor. Irradiation doses applied ranged from 10 to 1,000 Gy of gamma-rays. Hybrid selection was based on complementation of NR deficiency with wild-type NR genes. To aid in the analysis of somatic hybrids, species-specific repetitive DNA sequences from N. plumbaginifolia (NPR9 and NPR18) were cloned. NPR18 is a dispersed repetitive sequence occupying about 0.4% of the N. plumbaginifolia genome. In turn, NPR9, which is part of a highly repetitive DNA sequence, occupies approximately 3% of the genome. The species-specific plant DNA repeats, together with cytological analysis data, were used to assess the relative amount of the N. plumbaginifolia genome in the somatic hybrids. In fusion experiments using irradiated N. plumbaginifolia, an increase in irradiation dose prior to fusion led to a decrease in N. plumbaginifolia nuclear DNA content per hybrid genome. For some hybrid lines, an increase in the quantity of repetitive sequences was detected. Thus, hybrid lines 1NV/21, 100NV/7, 100NV/ 9, and 100NV/10 (where N. plumbaginifolia was the irradiated donor) were characterized by amplification of NPR9. In the reverse combination (where N. sylvestris was the irradiated donor), an increase in the copy number of NPR18 was determined for hybrid clones 1VC/2, 1VC/3, 100VC/2 and oct100/7. Possible reasons for the amplification of the repeated sequences are discussed.     

Mitochondrial DNA patterns are similar in gametosomatic and somatic hybrids of two Nicotiana species

Summary The segregation and recombination patterns of mitochondrial genome in the somatic hybrids of Nicotiana tabacum and N. rustica were studied by RFLP analysis using four heterologous mitochondrial DNA probes, namely cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), 26s rDNA and 5s-18s rDNA. These RFLP patterns were compared with those of the gametosomatic hybrids of these two species. A preponderance of N. rustica type patterns was observed in the somatic hybrids. One of the somatic hybrids had N. rustica type pattern with COI probe, novel pattern with COII, and 26s rDNA probe and N. tabacum type pattern with 5s-18s rDNA probe. These patterns are identical to those of some of the gametosomatic hybrids and could only be due to the recombination of mitochondrial genomes of the two parents. The extent and the nature of recombination of mitochondrial genomes is similar in gametosomatic and somatic hybrids.     

New Species of Cyst Nematode Heterodera pakistanensis (Nematoda: Heteroderidae) Attacking Wheat in Pakistan

Heterodera pakistanensis n. sp., described and illustrated from roots of common wheat (Triticum aestivum) from Sukkur, Sind, Pakistan, belongs to the goettingiana group. It is most closely related to H. cyperi Golden, Rau &Cobb, 1962, H. raskii Basnet & Jayaprakash, 1984, and H. mothi Khan &Husain, 1965. Second-stage juveniles (J2) can be distinguished from H. cyperi J2 by an areolated lateral field with four incisures and shorter stylet, whereas cysts are separated by a more elongated vulva slit and the conspicuous structure of the underbridge. It differs from H. raskii by having four areolated lateral lines in J2, smaller female lemon-shaped cyst, shorter fenestra length and width, conspicuous underbridge, and distinct anus with a high cuticular pattern 40-45 μm from posterior end. It also differs from H. mothi by the presence of four areolated lateral lines in J2 and absence of vulva denticles and bullae.     

Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species

Spicules of 9 Meloidogyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect-parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules of Xiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences.     

Influence of Herbicide Application to Soybeans on Soybean Cyst Nematode Egg Hatching

The hatching of Heterodera glycines eggs in soybean root exudates collected after postemergence application of three herbicides, and the hatching potential of H. glycines eggs from females feeding on herbicide-treated plants, were measured in vitro. Hatching in all root exudate solutions (RES) was greater than in deionized water but less than in 0.003 M ZnSO₄ solution. Filtering RES with a 0.22-μm-filter increased H. glycines hatching in RES. Application of acifluorfen, bentazon, and lactofen to foliage of soybean plants inhibited hatching of H. glycines eggs from the same plants. Hatching in RES from the different herbicide-treated soybeans was similar. Application of crop oil concentrate and non-ionic surfactant adjuvant to foliage did not affect hatching of H. glycines eggs from soybean plants.     

Genetic Analysis of Parasitism in the Soybean Cyst Nematode Heterodera Glycines

A genetic analysis of parasitic ability in the soybean cyst nematode Heterodera glycines was performed. To identify and characterize genes involved in parasitism, we developed three highly inbred H. glycines lines, OP20, OP25 and OP50, for use as parents for controlled crosses. Through these crosses, we have identified genes in the inbred parents that control reproduction of the nematode on hosts that carry resistance genes. These genes, designated as ror-* for reproduction on a resistant host, segregate in a normal Mendelian fashion as independent loci. Host range tests of F(1) generation progeny indicated that at least one parasitism gene in both the OP20 and OP50 lines for host PI 88788 was dominant. Parasitism genes in OP50 for hosts ``Peking' and PI 90763 are recessive. Two types of single female descent populations, a single backcrossed BC(1)F(2)-derived and a double backcrossed BC(2)F(1)-derived, were established on the susceptible soybean cultivar ``Lee 68.' Host range tests for parasitism in these lines demonstrated the presence of two independent genes in OP50, one for host PI 88788 designated ror-1 and one for host PI 90763 designated ror-2. OP20 carries two independent genes for parasitism on PI 88788, designated as alleles kr3 and kr4.     

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.     

Development of PrimeTime-Real-Time PCR for Species Identification of Soybean Cyst Nematode (Heterodera glycines Ichinohe, 1952) in North Carolina

Soybean cyst nematode (SCN) is an obligate, sedentary parasite that is a major pathogen of soybean and accounts for an estimated 1 billion dollars in production losses annually in the United States of America. This paper describes the development of a real-time PCR method for rapid, sensitive, species-specific and accurate identification of SCN alone or on mixed populations with other nematodes in North Carolina. The 83-bp DNA fragment of PrimeTime-real-time PCR was designed based on a 477-bp-SCN-SCAR marker previously proved to be SCN-specific. A total of 44 populations including cyst forming nematodes (Heterodera glycines, H. fici, H. schachtii, H. trifolii, Cactodera weissi, Globodera tabacum, Meloidodera floridensis and other unidentified cyst nematodes) and non-cyst forming nematodes (Ditylenchus dipsaci, Meloidogyne incognita and Xiphinema chambersi) were tested in this study, all SCN populations are tested positive and non-SCN populations negative. This assay for the detection and identification has been successfully applied for testing a single SCN cyst, a 2^nd-stage-SCN juvenile, a single SCN egg, up to ten SCN cysts, a 10-fold dilution of a single 2^nd-stage-SCN juvenile and 20-fold dilution of one SCN cyst. The assay is not SCN-race specific. It gave an accurate positive result when SCN is mixed with other cyst species. Also, nematode universal primers/probes for real-time PCR amplification as a nematode endogenous control to detect the presence of 18S ribosomal RNA (rRNA) gene were employed in this assay, so that a SCN-negative sample can be tested to exclude false negative. This method will be very useful for a broad range of research programs as well as the regulatory response and management of SCN in North Carolina and other region of the southeastern U.S.A.     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative