Genetic Uniformity Among Isolates of Peronospora tabacina, the Tobacco Blue Mold Pathogen

ABSTRACT As a first step toward analysis of genetic variation and population structure in Peronospora tabacina, we used a collection of random genomic DNA fragments to survey for restriction fragment length polymorphisms (RFLPs) in DNA from a collection of isolates from Kentucky and other tobacco-growing regions of the United States. Also included in the study were isolates from the wild tobacco species, Nicotiana repanda, and from ornamental tobacco, N. alata. In a preliminary survey using DNA from 10 pathogen isolates, no polymorphisms were detected at six single-copy DNA loci using 22 probe-enzyme combinations. Moderately repetitive and highly repetitive regions of the genome were also remarkably similar between isolates, with only 6 of 15 different probes identifying genetic differences. Some of the polymorphic probes were then used to analyze a larger collection of isolates, most of which were from Kentucky. This resulted in the identification of very few additional polymorphisms, indicating that the population of P. tabacina that infects the Kentucky tobacco crop is genetically very homogeneous. The low level of polymorphism detected in this study overall, suggests that genetic variability may be lacking in P. tabacina populations throughout the United States. Two of the RFLP markers gave hybridization patterns that were consistent with P. tabacina being diploid. Frequencies of alleles at these loci and linkage disequilibrium between different marker loci indicated that genetic recombination does not occur frequently in the pathogen population. DNA polymorphisms that were identified in this study enabled us to differentiate the pathogen population into at least 10 haplotypes. One isolate was analyzed in detail and was shown to be genetically stable through several rounds of single-spore isolation and through several pathogenic cycles.     

Single Cystosorus Isolate Production and Restriction Fragment Length Polymorphism Characterization of the Obligate Biotroph Spongospora subterranea f. sp. subterranea

ABSTRACT Spongospora subterranea f. sp. subterranea causes powdery scab in potatoes and is distributed worldwide. Genetic studies of this pathogen have been hampered due, in part, to its obligate parasitism and the lack of molecular markers for this pathogen. In this investigation, a single cystosorus inoculation technique was developed to produce large amounts of S. subterranea f. sp. subterranea plasmodia or zoosporangia in eastern black nightshade (Solanum ptycanthum) roots from which DNA was extracted. Cryopreservation of zoosporangia was used for long-term storage of the isolates. S. subterranea f. sp. subterranea-specific restriction fragment length polymorphism (RFLP) markers were developed from randomly amplified polymorphic DNA (RAPD) fragments. Cystosori of S. subterranea f. sp. subterranea were used for RAPD assays and putative pathogen-specific RAPD fragments were cloned and sequenced. The fragments were screened for specificity by Southern hybridization and subsequent DNA sequence BLAST search. Four polymorphic S. subterranea f. sp. subterranea-specific probes containing repetitive elements, and one containing single copy DNA were identified. These RFLP probes were then used to analyze 24 single cystosorus isolates derived from eight geographic locations in the United States and Canada. Genetic variation was recorded among, but not within, geographic locations. Cluster analysis separated the isolates into two major groups: group I included isolates originating from western North America, with the exception of those from Colorado, and group II included isolates originating from eastern North America and from Colorado. The techniques developed in this study, i.e., production of single cystosorus isolates of S. subterranea f. sp. subterranea and development of RFLP markers for this pathogen, provide methods to further study the genetic structure of S. subterranea f. sp. subterranea.     

Discrimination between Virulent and Attenuated Isolates of Tomato mosaic virus by Restriction Fragment Length Polymorphism

The three missense mutations on the gene for the 130-K protein of Tomato mosaic virus (ToMV) L₁₁A have been thought to be responsible for the attenuation of its virulence. The Eco47I RFLP detecting the missense mutation at 2349 successfully discriminated L₁₁A and its derivative attenuated isolates from ToMV virulent ones. RFLP analysis and mismatch amplification assay detecting the missense mutations at 1117 and 2754, respectively, could not discriminate some of the attenuated isolates from the virulent ones. These results indicated that, of the three missense mutations, only the one at 2349 was conserved in all the L₁₁A-derivative attenuated isolates. Received 16 March 2001/ Accepted in revised form 22 June 2001     

Genetic Analysis and Identification of Amplified Fragment Length Polymorphism Markers Linked to the alm1 Avirulence Gene of Leptosphaeria maculans

ABSTRACT A gene-for-gene interaction was previously suggested by mapping of a single major locus (LEM 1) controlling cotyledon resistance to Leptosphaeria maculans isolate PHW1245 in Brassica napus cv. Major. In this study, we obtained further evidence of a gene-for-gene interaction by studying the inheritance of the corresponding avirulence gene in L. maculans isolate PHW1245. The analysis of segregating F(1) progenies and 14 test crosses suggested that a single major gene is involved in the interaction. This putative avirulence gene was designated alm1 after the resistance locus identified in B. napus. Amplified fragment length polymorphism (AFLP) markers were used to generate a rudimentary genetic linkage map of the L. maculans genome and to locate markers linked to the putative avirulence locus. Two flanking AFLP markers, AC/TCC-1 and AC/CAG-5, were linked to alm1 at 3.1 and 8.1 cM, respectively. Identification of markers linked to the avirulence gene indicated that the differential interaction is controlled by a single gene difference between parental isolates and provides further support for the gene-for-gene relationship in the Leptosphaeria-Brassica system.     

Development of an artificial diet for mass rearing of the spiny bollworm,Earias insulana

An artificial diet for rearing the spiny bollworm,Earias insulana (Boisd.), was developed in experiments with three successive generations. The present recommended diet is based on kidney beans, alfalfa meal, whole powdered milk and yeast; methyl-P-hydroxy benzoate, chloramphenicol and formaldehyde were included as preservatives. The effect of the diets on the insect quality is discussed.     

水葫芦象甲的生物学及其寄主专一性

在室温 2 5～ 2 8℃的养虫室内 ,水葫芦象甲雌成虫寿命最长可达 1 6 1d ,最大卵量为 1 0 91粒。产卵期为 2 0～ 1 59d。卵主要产在叶柄内。幼虫孵化后在叶片内和叶柄中取食 ,并向植株基部钻蛀。幼虫期 30～ 4 0d。老熟幼虫在活的水葫芦植株根部化蛹 ,蛹期 2 5～ 30d。在温州自然条件下 ,象甲年发生 2代 ,并有明显的世代重叠现象。以成虫和幼虫越冬。利用 2 3科 4 6种植物进行的寄主专一性测定发现 ,该象甲只为害水葫芦 ,可安全用于水葫芦的生物防治     

转基因溶菌酶羊hLZ-J1品系特异性数字PCR方法及样本中痕量转基因成分检测

人溶菌酶是一种天然抗生素,具有抗细菌、真菌,抗炎症,预防感染等功能。转基因溶菌酶山羊可以在乳腺中特异性表达,从而可以从羊乳中规模化制备溶菌酶。本研究旨在建立一种适用于痕量样本中转基因溶菌酶羊成分定量检测的品系特异性数字PCR方法,并利用该方法对山羊的血液、羊奶、粪便和环境土壤样本等进行检测,用于对转基因溶菌酶山羊成分的溯源。建立的数字PCR方法具有高特异性、灵敏度、准确性,检测限和定量限低至15个拷贝每个反应,可以对羊血、羊乳等制品进行准确的定量检测。研究结果还表明该方法可以准确测定粪便和环境土壤样本中的痕量转基因溶菌酶羊成分,可进一步用于转基因动物外源基因的水平转移检测及环境风险评估。     

Effects of three management strategies on the seedbank, emergence and the need for hand weeding in an organic arable cropping system

The effects of three different weed management strategies on the required input of hand weeding in an arable organic farming system, the weed seedbank in the soil and the emerging weed seedling emergence were studied from 1996 to 2003. Strategies were based on population dynamic models and aimed for (1) control of weeds as carried out in standard organic farming practice, (2) control of all residual weeds that grow above the crop and (3) prevention of all weed seed return to the soil. Under all strategies, the size of the seedbank increased during the conversion from conventional to organic farming systems. The increase under strategy 3 was significantly smaller than the increase under the other strategies. From 1999 onwards, the weed densities in plots treated with strategy 3 became significantly lower than the weed densities in plots treated with the other strategies. The time needed for hand‐weeding required to prevent weed seed return, in addition to the time needed in standard organic farming practices, reduced during the course of the study. A management strategy aimed at the prevention of seed return (strategy 3) can reduce the size of the increase of the seedbank, which is usually observed after transition from conventional to organic farming. This study provides unique real‐world data that are essential for evaluating population dynamic models. The results may contribute to the development of weed management systems based on ‘no seed’ threshold strategies and to a further decrease in the dependence on herbicides.     

Testing of an integrated regime for effective and sustainable control of invasive Crofton weed (Ageratina adenophora) comprising the use of natural inhibitor species,activated charcoal,and fungicide

Crofton weed is a major invasive species in China. It exhibits superior growth characteristics and can outcompete with native species via allolepathic effects and modulation of the soil fungal microbiome. The simple removal of invading plants will not ensure restoration of the habitat due to the persistence of allelochemicals and viable seeds in the surrounding soil. An orthogonal experimental design was employed to evaluate the effects of three control factors (A, powdered natural inhibitor species to retard growth; B, activated charcoal to absorb allelochemicals; and C, fungicide to reduce fungal modulation effects), applied at three levels, on the growth and competitive ability of Crofton weed against two native species, in a pot‐culture experiment. All treatments reduced all measured growth parameters (P < 0.05) except for a specific leaf area, when compared with control plants. Furthermore, the competitive capacity of Crofton weed was decreased in the treatments while that of the native species was improved. Application to soil of the powdered natural inhibitor species and of activated charcoal significantly inhibited plant growth and competitive ability of Crofton weed (P < 0.05). Application of fungicide was less effective, but significantly reduced the specific leaf area of Crofton weed plants (P < 0.05). The specific combination of factors producing the greatest decrease in plant growth and competitive ability (compared with the control) included the addition of Delavaya toxocarpa powder (37.5 g per kg soil), addition of activated charcoal to soil at a ratio of 1:3 (v/v) (62.5 g per kg soil), and application of fungicide (Thiophanate‐Methyl) (0.28 g per kg soil).     

Development of a TaqMan probe-based insulated isothermal PCR (TiiPCR) for the detection of <Emphasis Type="Italic">Acidovorax citrulli</Emphasis>, the bacterial pathogen of watermelon fruit blotch

Watermelon (Citrullus lanatus) is an important crop of the Cucurbitaceae family in fruit production worldwide. During its production, bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Acidovorax avenae subsp. citrulli) is an important limiting factor on the volume and value of crops. This pathogen is known as a seed-borne pathogen, and the infested seeds can be a primary source of inoculum. Hence, a rapid and sensitive method for detecting A. citrulli on seeds would be an important tool in the management of BFB. In this study, we sought to develop a method to detect A. citrulli bacterial cells based on a TaqMan probe-based insulated isothermal PCR (TiiPCR) assay. Firstly, the specific primers and probe were designed based on a specific DNA fragment from the genome of A. citrulli. Then, PCR amplification was performed with the plasmid DNA to adjust the components of the PCR reagents, such as the concentrations of primers, magnesium chloride, and Taq DNA polymerase. Results revealed that 10 copies of plasmid DNA were detectable within the modified reagents by TiiPCR. Moreover, 10 bacterial cells in each reaction tube were detectable at a 100 % detection rate in this condition with a fluorescent signal intensification over 1.8. Based on these results, we concluded that a specific, rapid, and sensitive method based on TiiPCR had been successfully developed to detect bacterial cells of A. citrulli.