赤水河流域土壤放线菌及其拮抗活性研究

从贵州赤水河流域茅台镇及周边地区采集土样,用高氏1号培养基从6份土样中共分离到放线菌32株,根据形态学和生理生化特征,从中筛选15株进行分子鉴定和系统发育树分析,16S rDNA测序比对结果表明：MA06为白色类诺卡氏菌(Nocardioides albus),MA24为原小单孢菌(Promicromonospora),其余13株为链霉菌(Streptomyces),其中MA03为极暗黄链霉菌(Streptomyces fulvissimus),MA14为Streptomyces europaeiscabiei,MA07属Streptomyces sp.。通过构建系统发育树发现：15株放线菌分离株聚为三支,MA06和MA24各单列一支,其余的聚为第三大支,进一步表明了链霉菌属、类诺卡氏菌属和原小单孢菌属相互之间亲缘关系较远。抗菌活性检测发现M07、M25、M30和M32抗菌作用强,预示其作为生防资源菌和抗生素产生菌的潜在价值。     

斑马鱼迟缓爱德华氏菌的鉴定、致病性及药物敏感性

从患病斑马鱼(Danio rerio)肝脏组织分离到菌株Z1,通过对该菌株形态特征、生理生化特性、ATB系统鉴定和16S rRNA、DNA促旋酶B亚单位蛋白(gyrB)基因测序等综合分析,鉴定Z1菌株为迟缓爱德华氏菌(Edwardsiella tarda).Z1菌株对健康斑马鱼和透明四带无须鲃(Puntius tetrazona)人工感染试验发现,感染发病症状与自然发病症状一致,再分离菌株的特性与Z1菌株相同,并再次感染成功,证实迟缓爱德华氏菌为斑马鱼病原菌.药敏试验发现Z1菌株对呋喃妥因等11种药物敏感,对苯唑西林等6种药物耐受.15种中草药对Z1菌株的体外抑菌试验结果发现,五倍子、石榴皮的抑菌作用明显,最小抑菌质量浓度(MIC)和最小杀菌质量浓度(MBC)均≤6.25 mg/mL;艾叶等有一定抑菌作用,MIC和MBC均在25～200 mg/mL;而野菊花等抑菌作用不明显,MBC＞200 mg/mL.     

Characterization of Porcine Reproductive and Respiratory Syndrome Virus Deletion Mutant

The genome of the isolate of porcine reproductive and respiratory syndrome virus （PRRSV） from China, designated HPBEDV, was sequenced and analyzed. The size of the genome of HPBEDV was 15 334 nucleotides （nt）. Comparative analysis of HPBEDV with the genomic sequences of the domestic and other isolates （JXA 1, HUN4, CH-1 a, B J-4, VR2332, and LV） revealed that HPBEDV shared 98.4, 98.0, 89.0, 88.7, and 88.6% identity with the American strain JXA1, HUN4, CH- 1a, BJ-4, and VR2332, respectively, but only 54.7% identity with the European reference strain Lelystad virus. The NSP2 gene had 2 850 nt and encoded 950 amino acids （aa）, with two discontiguous deletions of 1 aa and 29 aa at positions 482 and 534-562, respectively, relative to VR-2332. Also, phylogenetic analysis with the published PRRSV genomic sequences indicated that the newly emerging isolate form a clade with the VR-2332 isolates. Therefore, HPBEDV was a novel strain with deletions in NSP2 gene.     

昆虫病原线虫共生细菌的分子鉴定及其培养特性的初步研究

【目的】确定昆虫病原线虫共生细菌YL001和YL002菌株的分类及其系统进化地位,并为该类生物资源的后续研究及开发应用奠定基础。【方法】对YL001和YL002菌株进行分子鉴定,PCR扩增并克隆其16SrRNA全序列,对其全序列进行了序列测定及系统进化分析,并采用参数测定法对其培养特性进行了初步研究。【结果】YL001和YL002菌株与嗜线虫致病杆菌(Xenorhabdus nematophila)种内菌株形成一个类群,序列同源性大于99%,与发光杆菌(Photorhabdus)属内菌株的序列同源性大于94%。2菌株的延缓期、对数生长期、稳定期和衰亡期分别为0~6,6~18,18~66和66 h;培养12 h后,YL001和YL002菌株发酵液的pH值分别降低至5.70和5.56,此后逐渐上升,至发酵结束时其pH值分别为7.74和8.07;培养0~18 h时2菌株发酵液中还原糖含量迅速降低,此后保持稳定;12 h时氨基氮含量达到最低,此后开始缓慢上升;YL001菌株培养54 h后及YL002菌株培养42和66 h后,其对番茄灰霉病菌和枯草芽孢杆菌的抑制作用最强。【结论】YL001和YL002菌株属于嗜线虫致病杆菌种内的菌株;YL001和YL002菌株的培养特性符合细菌生长和代谢的一般规律,其适宜的发酵周期分别为54和66 h。     

藏獒线粒体DNA D-loop区序列测定及其分类地位研究

根据家犬线粒体基因组DNA序列设计引物,获得了藏獒(Tibetan mastiff)线粒体DNA D-loop区全序列,并以灰狼、郊狼作为外类群对藏獒、家犬亚种内6个大型家犬品种进行了系统发育分析.试验结果发现:藏獒线粒体DNA D-loop区序列全长1 250 bp,与家犬源序列的同源性为96.06%,其中有20个碱基缺失;系统发育分析发现藏獒、家犬亚种内6个大型家犬品种与灰狼聚为一大类,而郊狼自成一类,说明藏獒与其他家犬品种一样起源于灰狼,是家犬亚种内的一个品种,在分类地位上隶属于食肉目、犬科、犬属、狼种、家犬亚种;在家犬亚种内,藏獒与英国牧羊犬、兰伯格犬聚为一类,而德国牧羊犬与瑞典猎鹿犬、黑俄罗斯更犬聚在一起,再与圣伯纳犬聚为一类,从分子水平上验证了英国牧羊犬等大型犬可能含有藏獒血统的观点.     

鳗鲡病原菌16SrRNA基因序列测定及系统进化分析

对从福建省不同鳗鲡养殖场发病鳗鲡肝脏中分离并经感染证实的35株致病菌的16S rRNA基因进行序列测定和系统进化分析。用CTAB法提取各菌株DNA,以细菌16S rDNA通用引物进行PCR扩增,用限制性片段长度多态性分析1、6S rDNA序列分析和系统进化分析对其进行分子鉴定,构建系统进化树。结果表明:35株致病菌分别属于γ-变形菌纲和厚壁菌门2大类群的6个属和1个科,主要种类是气单胞菌属的嗜水气单胞菌、维氏气单胞菌、简氏气单胞菌和豚鼠气单胞菌等,占63%;其次是芽孢杆菌属细菌;少量为鲁氏耶尔森菌、弗氏柠檬酸杆菌、克雷伯氏菌属、假交替单胞菌属细菌和肠杆菌科细菌。     

Isolation and Molecular Characterization of Fowl Adenovirus and Avian Reovirus from Breeder Chickens in Japan in 2019–2021

Fowl adenoviruses (FAdVs) and avian reoviruses (ARVs) are ubiquitous in poultry farms and most of them are not pathogenic, yet often cause damage to chicks. A total of 104 chicken fecal samples were collected from 7 farms of breeder chickens (layers and broilers) in Japan from 2019 to 2021, and yielded 26 FAdV plus 14 ARV isolates. By sequencing, FAdV isolates were classified as FAdV-1, 5 and 8b. ARV isolates were classified as genotype II, IV and V. These results suggest that FAdVs and ARVs are resident in the breeder chicken farms in Japan.     

Characteristics and distribution of Colletotrichum species in coffee plantations in Hainan,China

Anthracnose caused by Colletotrichum species is a serious disease on a range of economically important hosts. To determine the Colletotrichum species in coffee plantations in Hainan, China, 55 isolates were obtained from Coffea arabica (arabica) and C. canephora var. robusta (robusta) in five counties. Initially, partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to measure fungal genetic diversity. Then a subset of 23 isolates was selected to represent the range of genetic diversity, varieties and geographic origin for further multilocus phylogenetic analyses. These isolates belong to eight known Colletotrichum species from three Colletotrichum species complexes, including gloeosporioides (C. endophytica, C. fructicola, C. ledongense, C. siamense and C. tropicale), boninense (C. karstii), gigasporum (C. gigasporum), and one singleton species (C. brevisporum). Of these, C. siamense was isolated in all sampled counties and C. fructicola was identified in three counties. The other six species were isolated only in one or two counties. Only C. siamense and C. fructicola were isolated from arabica, whereas all eight species were isolated from robusta. Occurrence of C. brevisporum, C. endophytica, C. ledongense and C. tropicale in coffee has not been reported previously. Pathogenicity tests showed that all eight species were pathogenic to coffee leaves and fruit. In vitro tests showed that Colletotrichum isolates from coffee in Hainan were most sensitive to prochloraz, less sensitive to carbendazim, propiconazole and difenoconazole, and least sensitive to myclobutanil.     

Mating system complexity and cryptic speciation in the seed bank pathogen Pyrenophora semeniperda

Mating strategies contributing to a balance between inbreeding and outcrossing are understudied in all but a few model fungal pathogens. This study examined factors that influence the occurrence of the sexual state of Pyrenophora semeniperda. It was consistently found to have functional copies of the MAT1-1 and MAT1-2 idiomorphs essential for sexual reproduction, despite considerable polymorphism in both single nucleotide polymorphisms (SNPs) and number of 18-base minisatellite repeats. The two idiomorphs occurred at approximately equal frequencies across 25 populations on Bromus tectorum seeds in the western United States, suggesting maintenance of sexual reproduction. The putative mating system is described as facultative pseudohomothallism, with only one of the two MAT1 idiomorphs found in a nucleus. Unikaryotic strains of opposite mating type can potentially mate, as can nuclei of opposite mating type in a thallus that results from anastomosis between vegetatively compatible unikaryotic strains. Strains shown to be dikaryotic using simple sequence repeat (SSR) markers may contain either or both MAT1 idiomorphs. Most populations consist of a mixture of MAT1-1, MAT1-2 and MAT1-1/MAT1-2 genotypes. A possible constraint on recombination is the presence of multiple strain groups characterized by ITS haplotype. These are apparently vegetatively incompatible, as even dikaryotic strains are invariably composed of a single ITS haplotype. Different ITS haplotypes also have unique combinations of MAT1-1 and MAT1-2 allelic variants, suggesting that perhaps these strain groups are also sexually incompatible. Phylogenetic analysis using both genome-wide SNP/indel polymorphisms and SSR markers demonstrated genetic divergence among ITS haplotypes, supporting the hypothesis that these strain groups may represent cryptic species.     

Geographic location and year determine virulence,and year determines genetic change,in populations of Neopseudocercosporella capsellae

White leaf spot (Neopseudocercosporella capsellae) can be severe and problematic worldwide across both horticultural and oilseed Brassicaceae, including susceptible rapeseed. In this study, 82 isolates from 2015 and 106 isolates from across Australia in 2016 were first assessed for their virulence against three different rapeseed (Brassica napus) cultivars. For both years there were significant (P < 0.001) differences. Also, there were significant (all P < 0.001) differences between isolates in each year, and between cultivars. For 2016 isolates, there were also significant differences (P < 0.001) between isolates across three different Australian states, and a significant interaction (P < 0.001) between isolates with cultivars. Of the three Australian states, isolates from Victoria were most virulent. Among tested cultivars, cv. Scoop was most susceptible. Subsequently, phylogenetic analysis of 114 of these same 2015 and 2016 isolates showed current isolates clustered separately from the majority of 2005 N. capsellae isolates collected from Western Australia a decade earlier, confirming significant genetic change within N. capsellae populations over the past decade. However, isolate clusters showed no association with geographical location. The results suggest that phylogenetic association among 2005 and 2015–2016 N. capsellae isolates is complementary with pathogenicity variations explained by geographically different N. capsellae pathogen populations. Neopseudocercosporella capsellae populations are evolving rapidly, challenging management through host resistance at a time of increasing incidence and severity of white leaf spot disease over the past decade. The outcome is well illustrated by cv. Scoop, previously resistant to 2005 isolates but moderately susceptible to 2015 and highly susceptible to 2016 isolates.