Molecular phylogenetics and microsatellite analysis reveal a new pathogenic Ceratocystis species in the Asian‐Australian clade

The ascomycete genus Ceratocystis has a broad geographic distribution and includes pathogens of a wide range of mostly woody hosts. Black rot of Colocasia esculenta (taro), a popular cultivated root crop in China, is caused by a species of Ceratocystis broadly treated as C. fimbriata sensu lato. Recently, isolates of Ceratocystis were obtained from black rot lesions on C. esculenta corms in two Chinese provinces. Sequence comparison of the ITS, partial β‐tubulin, TEF‐1α, MS204 and RPBII gene regions were used to identify these isolates and compare them to other Ceratocystis species. Furthermore, the diversity of Ceratocystis species in the Asian‐Australian clade (AAC) was investigated using 23 microsatellite markers. Results showed that the isolates from C. esculenta represent a novel species, which is morphologically and phylogenetically different to closely related species in the AAC, and is described here as Ceratocystis changhui sp. nov. In microsatellite analyses, this new species also emerged as distinct from others in the AAC. Inoculation tests showed that C. changhui sp. nov. is a virulent pathogen of C. esculenta corms, but produces only small lesions on Eucalyptus grandis and Eriobotrya japonica.     

Effect of inhibitors of melanin biosynthesis on appressorial penetration and reductive reactions in Pyricularia oryzae and Pyricularia grisea

Appressorial penetration of onion epidermal walls by wild-type strains P-2 and O-42 of Pyricularia oryzae was more sensitive to fthalide (4,5,6,7-tetrachlorophthalide) than penetration by wildtype strain 4091-5-8 and black mutant strain BL-3 of Pyricularia grisea. Cerulenin completely blocked appressorial penetration by P. oryzae strain P-2 or P. grisea strain BL-3, but penetration capacity of these appressoria could be largely restored with 0.1 mM scytalone. Fthalide and tricyclazole inhibited the conversion of 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN) to 1,3,8-trihydroxynaphthalene (1,3,8-THN) and 1,8-dihydroxynaphthalene (1,8-DHN) as well as the conversion of scytalone to 1,8-DHN by cell-free extracts of P. oryzae or P. grisea. These inhibitors blocked the NADPH-dependent reductase reactions involved in the conversion of 1,3,6,8-THN to scytalone and the conversion of 1,3,8-THN to vermelone in the melanin biosynthetic pathway to 1,8-DHN. The two reductase reactions in extracts from P. oryzae P-2 were about 10 times as sensitive to fthalide or tricyclazole as those in extracts of P. grisea. Reductase activity with either 1,3,6,8-THN or 1,3,8-THN as substrates was present in only trace amounts in cell-free extracts of the buff mutant, P. oryzae P-2 m-1.     

旱地小麦产量及其构成因素灌溉效应的模拟分析

为揭示灌水时期与灌水量对黄土丘陵区小麦产量及其构成因素的影响规律,依据黄土丘陵典型区域定西1971-2016年气象数据及定西市安定区凤翔镇安家沟村2016年大田试验数据,利用APSIM（Agricultural Production Systems Simulator）平台,动态模拟各灌溉处理下的小麦生长发育过程,并对各灌溉响应进行模拟分析。结果表明：小麦全生育期需水量为315.25 mm,各生育阶段需水量为播种~出苗21.13 mm、出苗~分蘖26.60 mm、分蘖~拔节33.27 mm、拔节~孕穗77.17 mm、孕穗~开花69.31 mm、开花~灌浆36.19 mm、灌浆~成熟51.58 mm。为提高产量,最佳灌水时期应在播种至拔节,灌水量不宜超过60 mm;开花前各生育阶段灌溉处理都明显提高了单位面积籽粒数量,其中以分蘖~拔节阶段（TS）最明显,拔节~孕穗阶段（JS）灌水量不宜超过80 mm、孕穗~开花阶段（BS）灌水量不宜超过20 mm。开花~灌浆阶段（FS）灌水量40 mm对粒重增加的促进作用最明显。     

Molecular characterization and experimental host range of Euphorbia mosaic virus-Yucatan Peninsula, a begomovirus species in the Squash leaf curl virus clade

Euphorbia mosaic virus (EuMV), a tentative species within the genus Begomovirus, was isolated from Euphorbia heterophylla plants growing in the Yucatan Peninsula, Mexico. The complete bipartite genome was cloned from total DNA extracts and the nucleotide (nt) sequence was determined. The DNA-A sequence of the EuMV-Yucatan Peninsula (EuMV-YP) isolate shared 95% nt identity with the partially characterized type EuMV isolate from Puerto Rico. The EuMV-YP genome organization was like that of other New World, bipartite begomoviruses. The DNA-A component was 2613 nt in size, while the DNA-B component was 2602 nt long. The 165-nt common region (CR) sequence for the DNA-A and DNA-B components shared a lower than expected nt identity of 86%. The organization and iterons of the putative AC1 binding site of EuMV-YP were similar to those of begomoviruses in the Squash leaf curl virus (SLCV) clade. Characteristic disease symptoms were reproduced in E. heterophylla plants inoculated at the seedling stage using the cloned viral DNA-A and DNA-B components, confirming disease aetiology. Results of an experimental host-range study for EuMV-YP indicated that it infected at least five species in three plant families, including the Euphorbiaceae ( E. heterophylla ), Solanaceae ( Datura stramonium , pepper, tomato) and Fabaceae (bean). Phylogenetic analysis of the DNA-A and DNA-B components indicated that EuMV-YP is a New World begomovirus and that it is a new member of the SLCV clade.     

Pathogenic Variability of Pyricularia grisea from the High- and Mid-Elevation Zones of Bhutan

ABSTRACT Thirty isolates of P. griseacollected from rice during a blast epidemic in 1995 in the high (1,800 to 2,600 m) and middle (1,200 to 1,800 m) elevations of Bhutan and 80 isolates collected from one rice cultivar from two high- and two mid-elevation sites in 1996 were analyzed for virulence. Differential varieties were indica CO39, with five near-isogenic lines (NILs) for resistance genes in the genetic background of CO39, and japonica Lijiangxintuanheigu (LTH), with five NILs for LTH. Twelve selected Bhutanese landraces also were studied. In addition, 10 blast nurseries consisting of the NIL sets, important local landraces, and representatives of international differential groups were established in the 1996 and 1997 growing seasons in the mid- and high-elevation agroecological zones. The 110 isolates were differentiated into 53 pathotypes based on the 2 NIL sets. Thirteen isolates were avirulent on all of the NILs but were compatible with some landraces. Several isolates were able to attack one of the NILs of CO39 but not CO39. These results strongly suggest that both CO39 and LTH possess previously unidentified resistance. The landraces were not uniform in their reactions to the isolates. When a reaction index taking into account all individual plant reactions was used, isolates that had been assigned to the same pathotype could be further differentiated, indicating that the NIL sets could not completely discriminate virulences in Bhutanese P. grisea populations. In the trap nurseries, disease was always present in the middle elevations, but disease was very low during July 1996 in the high elevations and only present during August and September 1997. Almost all varietal groups were more frequently attacked in the middle than in the high elevations, indicating that the virulence spectrum is wider and the conduciveness of the environment is greater in the middle elevations. Landraces from the high elevations were most susceptible, followed by international differential groups 7 and 8. The results suggest that selection has yielded landraces with more complete and complex resistance in the more disease-conducive mid-elevation environment. At the same time, the pathogen population also possesses a wider virulence spectrum in that environment.     

进口木材中碎木长蠹属3个近似种的鉴别

2009年3月,深圳出入境检验检疫局从进口非洲多哥的原木中检出在我国未有分布记载的林木重要害虫--棋盘碎木长蠹[Xyloperthlla scutula(Lesne)].本文介绍了近年从进口原木、板材中截获的3种碎木长蠹:多毛碎木长蠹[X.crinitarsis(Imhoff)]、脂松碎木长蠹[X.picea(Olivier)]和棋盘碎木长蠹,分别介绍了雌雄成虫的形态特征,并列出了这3个近似种的鉴别特征检索表.     

2002年江苏省水稻稻瘟病菌致病性及遗传多样性研究

 由稻瘟病菌(Magnaporthe grisea,无性世代为Pyricularia grisea)引发的水稻稻瘟病是目前水稻生产上的重要病害之一,存在着品种专化性和致病性的分化.一些抗病品种推广3~5年后即丧失抗病性,给生产带来很大危害.目前选育和利用抗病良种仍是防治该病最经济有效的措施^[1].由于稻瘟病菌的多样性及易变性直接导致了品种的抗病性丧失,因此研究稻瘟病菌的致病性和群体遗传结构组成等具有重要意义.     

Molecular and serological characterization of iris yellow spot virus, a new and distinct tospovirus species

ABSTRACT A new tospovirus was identified in iris cultivations in the Netherlands. Both serological comparisons and sequence determination of the S RNA demonstrate that this virus represents a new and distinct species, belonging to a separate serogroup, and for which the name iris yellow spot virus (IYSV) is proposed. The disease symptoms on iris are characterized by yellow spots on the leaves. Its experimental host range is very narrow and, in addition to iris, only includes Nicotiana benthamiana and Datura stramonium. The nucleoprotein of IYSV shows only 30 to 44% sequence identity with those of other tospoviruses identified so far; the highest homology being found with the tospovirus species of serogroup IV.     

稻瘟菌TAC文库的构建与评价

 本文构建了一个包含16 128个克隆的稻瘟菌基因组的TAC文库,74%的克隆含有外源插入片段,其插入片段大小平均为59 kb,该库相当于稻瘟菌基因组的18倍。本文并对文库的代表性进行了评价,采用3个单拷贝的标记作为探针筛库,分别得到16,17和16个阳性克隆,这与估算的该库的覆盖率是一致的;从MH 18S1为探针所筛选的阳性克隆中,随机挑取8个并对其限制性酶切图谱进行分析,结果表明:其组成的重叠群跨度为153 kb,未发现嵌合和缺失现象。以上结果表明,此TAC文库适合用于稻瘟菌基因组的分析。     

Taxonomic characterization of Pyricularia isolates from green foxtail and giant foxtail, wild foxtails in Japan

Twenty-eight Pyricularia isolates from two wild foxtails—green foxtail (Setaria viridis) and giant foxtail (S. faberii)—in Japan were taxonomically characterized by DNA analyses, mating tests, and pathogenicity assays. Although most of the isolates failed to produce perithecia in mating tests with Magnaporthe oryzae, a diagnostic polymerase chain reaction-restriction fragment length polymorphism phenotype of M. oryzae was detected in the beta-tubulin genomic region in all isolates. The pathogenicity assays revealed that host ranges of the isolates were similar to those of isolates from foxtail millet (S. italica), which were exclusively pathogenic on foxtail millet. In addition to the 28 isolates from wild foxtails, 22 Pyricularia isolates from 11 other grasses were analyzed by RFLP using single-copy sequences as probes. In a dendrogram constructed from the RFLP data, isolates that were previously identified as M. oryzae formed a single cluster. All the wild foxtail isolates formed a subcluster with foxtail millet isolates within the M. oryzae cluster. From these results, we conclude that Pyricularia isolates from the wild foxtails are closely related to isolates from foxtail millet and should be classified into the Setaria pathotype of M. oryzae.     

稻瘟病拮抗菌NK413的鉴定及抑菌效果

菌株NK413是从黑龙江省农田土壤中分离得到的一株放线菌,为明确其分类地位及在植物病害生物防治中的应用潜力,采用多相分类法对其进行鉴定,并采用孢子萌发抑制法、菌丝生长速率法、杯碟法考察了NK413菌株及其代谢产物对稻瘟病菌的抑菌效果及作用方式。结果表明:菌株NK413的形态特征、生理生化特征与小白链霉菌(Streptomyces albulus)接近,细胞壁类型为Ⅰ型、磷脂脂肪酸成分符合链霉菌属特征。16S rDNA全长为1 614 bp,与小白链霉菌亲缘关系最近,因此将NK413定名为小白链霉菌(S.albulusNK413)。NK413对稻瘟病菌孢子萌发和菌丝生长的抑制作用较强,原液对稻瘟病菌的抑菌圈直径达30 mm,无菌发酵滤液稀释1倍对孢子萌发的抑制率为100%,稀释5倍时对菌丝生长的抑制率为100%。抗菌谱广,具有较好的开发前景。     

Effect of tricyclazole on growth and secondary metabolism in Pyricularia oryzae

Tricyclazole [5-methyl-1,2,4-triazolo (3,4-b)-benzothiazole] controls rice blast disease caused by Pyricularia oryzae at concentrations (5–10 μg/ml) which do not inhibit growth of the pathogen in vitro. However, concentrations of 1 μg/ml or less inhibit melanin formation in the fungus. Production of pyriculol by the pathogen is usually enhanced by 10 μg/ml of tricyclazole, whereas production of 3,4-dihydro-4,8-dihydroxy-1(2H)-naphthalenone is strongly inhibited or markedly reduced and delayed. Evidence suggests that tricyclazole blocks aspects of polyketide metabolism in P. oryzae which may have a role in pathogenicity.     

Brazilian Potato virus Y isolates identified as members of a new clade facilitate the reconstruction of evolutionary traits within this species

Potato virus Y (PVY) is a plant virus distributed worldwide that causes damage to several species of the Solanaceae family. It was established long ago that groups of PVY isolates defined by phylogenetic analyses correlate strongly with those demarcated by differential biological properties. Consequently, life‐history traits of this viral species can be inferred by phylogenetic analysis. In this study, characteristics of PVY isolates sampled in different tobacco fields in Brazil were analysed and most of the tested Brazilian PVY isolates were assigned to the recently described unconventional serogroup Y^U. The analysis of molecular diversity of the coat protein (CP) cistron from some Y^U isolates made it possible (i) to identify specific amino acid residues in the N‐terminal of the CP protein and (ii) to assign some Y^U isolates to a new PVY clade. The symptoms caused by isolates belonging to this new PVY ‘Brazilian’ clade and their ability to infect selected susceptible hosts led to the conclusion that neither veinal necrosis symptoms expressed on infected tobacco plants nor adaptation to potato or pepper hosts are ancestral characteristics of PVY. These observations suggest that PVY has gained a remarkable new biological property and broadened its host range over time.     

Genetic Analysis of Avirulence/Virulence of an Isolate of Magnaporthe grisea from a Rice Field in Texas

ABSTRACT An isolate of Magnaporthe grisea, Tm4, from a rice field in Texas was crossed with a fertile laboratory strain, 70-6. The progenies showed segregation of avirulence/virulence on rice cvs. Newbonnet, Lemont, Lebonnet, Leah, and Katy. The avirulent/virulent segregation ratios were 29:6 on Newbonnet, Lemont, and Lebonnet; 28:7 on Leah; and 33:2 on Katy. There was cosegregation on the first three cultivars. Several avirulent progenies were backcrossed to virulent parent 70-6. Three generations of backcrossing avirulent progenies to 70-6 led to segregation ratios that suggested certain strains had only one avirulence gene. Strains avirulent only on cv. Katy or only on cvs. Newbonnet, Lemont, and Lebonnet were test crossed with virulent siblings. Strains that gave progeny ratios approximating 1 avirulent:1 virulent when crossed with virulent siblings were selected for further test crossing. Intercrosses between strains with possible single avirulence genes were made to determine whether these strains had the same or different avirulence genes. Many lines still segregated two genes for avirulence after three generations of backcrossing. This is based on the recovery of virulent progenies from crossing two avirulent siblings.     

Genetic differentiation of Magnaporthe oryzae populations from scouting plots and commercial rice fields in Korea

A previous study of the diversity and population structure of the rice blast fungus, Magnaporthe oryzae, over a 20-year period in Korea, found novel fingerprint haplotypes each year, and the authors hypothesized that populations might experience annual bottlenecks. Based on this model, we predicted that M. oryzae populations would have little or no genetic differentiation among geographic regions because rice blast is commonly found throughout Korea each year and M. oryzae would have to disperse from small populations surviving annually between rice crops. To test this hypothesis, we sampled M. oryzae from rice fields in eight provinces in Korea in a single year (1999). In four provinces, we sampled from a set of rice cultivars commonly grown in commercial fields (group I); because of low disease incidence in four other provinces, we could not sample from commercial fields and instead sampled from scouting plots of different cultivars set up for detecting new pathotypes of M. oryzae (group II). All isolates were genotyped with DNA fingerprint probes MGR586 and MAGGY, a telomere-linked gene family member TLH1, the PWL2 host specificity gene and mating type. Fingerprint haplotypes clustered into two distinct lineages corresponding to the two sets of cultivars (groups I and II), with haplotype similarities of 71% between lineages and >76% within lineages. Isolates from the same cultivar within group I were genetically differentiated among locations, and isolates within the same location were differentiated among cultivars. Differentiation for TLH1 and PWL2 was significant (P < 0.03), but not as strong as for fingerprint markers. Similar analyses were not possible among group II isolates because too few isolates were available from any one cultivar. All isolates were in the same mating type, Mat1-1, ruling out sexual reproduction as a source of novel haplotypes. When the 1999 samples were compared with the historical samples from the previous study, haplotypes of group I formed a separate cluster, while those of group II clustered with haplotypes from the historical sample. Altogether, geographic subdivision, monomorphism of mating type, and correlation of haplotypes to sets of cultivars are not consistent with the hypothesis of repeated turnover of haplotypes. Instead, the previous correlations of haplotypes to year might have been caused by inadequate sampling of haplotypes each year, highlighting the need for studies of population genetics to be conducted with systematic samples collected to address specific questions.     

广西稻瘟病和纹枯病的发生与综合防治

广西水稻年种植面积约3300万亩,近年水稻病虫发生为害呈逐年加重态势,年发生面积达9000万亩次,总体发生程度达4(5)级,尤其是稻瘟病和纹枯病的发生,给水稻造成了巨大的损失。稻瘟病是广西水稻上发生为害仅次于水稻纹枯病的病害,全区总体发生程度一般为中等,局部中等偏重发生,近几年年发生面积在800多万亩次左右,约占水稻种植面积的1/4多。其发生特点是:(1)以历史病区和种植感病品种的稻区,特别是桂东北、桂东南及沿海地区局部稻田发生较重;(2)个别感病的优质稻、常规稻发病后,往往易造成落窝,产量损失较大;     

云南元阳哈尼梯田稻瘟病菌遗传多样性分析

 利用12对SSR引物,对来自云南元阳哈尼梯田不同海拔高度、不同水稻品种上的稻瘟病菌菌株进行遗传多样性分析,并运用日本清泽建立的鉴别品种对其中62个菌株进行生理小种鉴定。结果显示:稻瘟病菌总体遗传多样性水平较高(He=0.57±0.27,I=1.17±0.60)。聚类分析表明,在80%相似水平下菌株可划分为9个多样性群体。供试菌株划分为16个生理小种,其中002、000和006为优势小种。分离的不同海拔稻瘟病菌生理小种和遗传多样性存在差异。海拔1600~1700m生理小种最丰富,达9个生理小种;海拔1500~1600m、1400~1500m和1700~1800m,生理小种分别为6、5和4个;遗传多样性分析表明,从海拔1400~1500m分离的菌株遗传多样性水平最高(He=0.57±0.24),并划分为6个群体。综合分析表明,在元阳哈尼梯田系统中,栽培传统品种遗传多样性越丰富的区域,其稻瘟病菌群体多样性和生理小种也越丰富。     

Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.