野生大豆资源对大豆疫病抗病性和耐病性鉴定

大豆疫病是大豆重要病害之一,在世界范围内导致严重经济损失。防治大豆疫病最有效方法是利用抗病或耐病品种。筛选抗性资源是发掘抗性基因和抗病育种的基础。本研究鉴定了野生大豆资源对大豆疫病的抗病性和耐病性,以期发掘优异抗源。苗期用子叶贴菌块方法鉴定104份野生大豆资源对两个不同毒力的大豆疫霉分离物PSJS2(毒力型:1a,1b,1c,1d,1k,2,3a,3b,3c,4,5,6,7,8)和PS41-1(毒力型:1a,1d,2,3b,3c,4,5,6,7,8)抗性,结果表明33份资源抗PS41-1,35份资源抗PSJS2,其中18份抗两个分离物。在抗病性鉴定基础性上,用菌层接种方法对选择的82份资源进行耐病性鉴定,发现7份高耐病性资源。这些结果表明,野生大豆中可能含有新的大豆疫病抗病和(或)耐病资源,这些抗病或耐病资源可以用于未来大豆抗病育种,以丰富大豆对大豆疫病的抗性遗传基础。     

Diversity among Single Zoospore Isolates Derived from Single-zoosporangia of Phytophthora sojae Kauf. and Gerd

Phytophthora sojae Kauf. and Gerd, a host specific pathogen to soybean, causes pre and postemergence damping-off and root and stem rot on soybean. The pathogen evokes severe yield losses in most soybean growing areas worldwide. The objective of this study was to determine phenotypic and genotypic diversity among single zoospore isolates (SZIs) originating from two single zoosporangia (Ps411-1 and Ps411-2) derived from the same parental isolate of P . sojae Ps411. Results showed that colony morphology and growth rate of 32 SZIs derived from sporangium Ps411-1 and 35 SZIs released from sporangium Ps411-2 did not significantly differ from the parental isolate Ps411. Pathogenicity of the SZIs was tested on three resistant and three susceptible Chinese soybean cultivars. While the majority of SZIs derived from sporangium Ps411-1(59.4%) and sporangium Ps411-2(71.4%) retained the same virulence spectrum as the parental isolate, the other SZIs of both progenies demonstrated either a higher or a lower level of virulence compared to that of parental isolate. A low level genetic variability in the populations of both single zoospore progenies was also demonstrated using the sequence-related amplified polymorphism (SRAP) technique. Cluster analysis separated the SZIs from both zoosporangia, Ps411-1 and Ps411-2, into four and three SRAP groups, respectively. No close correlation among SRAP and virulence could be established among SZIs. The results of this study suggest that virulence variability may be regarded as part of the total genetic changes among the zoospore progenies derived from single-zoosporangia. The pathogenic variability during asexual reproduction may play a role in changing the virulence structure of P . sojae .     

黑龙江省主要栽培大豆品种(系)对大豆疫霉根腐病的多抗性评价

用下胚轴伤口接种方法接种鉴定黑龙江省60个栽培大豆品种和育成品系对5个具有不同毒力大豆疫霉菌菌株41-4、PMCl、USAR4、PSZJ6和USAR17的抗性.有50个品种(系)抗1个或1个以上茵株或表现中间类型,其中有5个、8个、16个和21个品种(系)分别对4个、3个、2个和1个菌株表现抗性或中间类型.60个品种(系)对5个菌株共产生12种反应模式,其中呈RRSSR反应类型的品种(系)可能含有Rpslα或Rpslc基因,品系农大3861可能含有Rps3c基因,呈SSSSS反应模式的品种(系)可能含有Rps7基因,或不含抗病基因;其它9种反应模式与含有已知单基因品种或单基因组合的反应模式不同,可能具有未知抗病基因.该研究结果表明,黑龙江省具有较丰富的抗大豆疫霉根腐病大豆品种(系),大部分品种(系)的抗性是有效的,可合理地用于大豆生产和抗疫霉根腐病育种.     

Fusarium-produced vitamin B6 promotes the evasion of soybean resistance by Phytophthora sojae

Plants can be infected by multiple pathogens concurrently in natural systems. However,pathogen–pathogen interactions have rarely been studied. In addition to the oomycete Phytophthora sojae, fungi such as Fusarium spp. also cause soybean root rot. In a 3-year field investigation, we discovered that P. sojae and Fusarium spp. frequently coexisted in diseased soybean roots. Out of 336 P. sojae–soybean–Fusarium combinations,more than 80% aggravated disease. Different Fusarium species all enhanced P...     

抗大豆疫霉根腐病野生大豆资源的初步筛选

由大豆疫霉菌引起的大豆疫霉根腐病是严重影响大豆生产的毁灭性病害之一.防治该病唯一经济、有效和环境安全的方法是利用抗病品种.本研究对野生大豆资源进行抗大豆疫霉根腐病初步筛选,以期探讨野生大豆的抗性水平、分布和获得抗性野生大豆资源.通过苗期接种大豆疫霉菌对412份野生大豆资源进行抗病性鉴定,有13.4%的资源抗大豆疫霉根腐病,15.3%的资源表现为中间反应类型.对野生大豆资源的来源分析表明,抗大豆疫霉根腐病野生大豆资源在我国分布广泛,其中安徽省野生大豆资源抗性最丰富.     

Discriminant haplotypes of avirulence genes of Phytophthora sojae lead to a molecular assay to predict phenotypes

The soybean–Phytophthora sojae interaction operates on a gene-for-gene relationship, where the product of a resistance gene (Rps) in the host recognizes that of an avirulence gene (Avr) in the pathogen to generate an incompatible reaction. To exploit this form of resistance, one must match with precision the appropriate Rps gene with the corresponding Avr gene. Currently, this association is evaluated by phenotyping assays that are labour-intensive and often imprecise. To circumvent this limitation, we sought to develop a molecular assay that would reveal the avirulence allele of the seven main Avr genes (Avr1a, Avr1b, Avr1c, Avr1d, Avr1k, Avr3a, and Avr6) in order to diagnose with precision the pathotypes of P. sojae isolates. For this purpose, we analysed the genomic regions of these Avr genes in 31 recently sequenced isolates with different virulence profiles and identified discriminant mutations between avirulence and virulence alleles. Specific primers were designed to generate amplicons of a distinct size, and polymerase chain reaction conditions were optimized in a final assay of two parallel runs. When tested on the 31 isolates of known virulence, the assay accurately revealed all avirulence alleles. The test was further assessed and compared to a phenotyping assay on 25 isolates of unknown virulence. The two assays matched in 97% (170/175) of the interactions studied. Interestingly, the sole cases of discrepancy were obtained with Avr3a, which suggests a possible imperfect interaction with Rps3a. This molecular assay offers a powerful and reliable tool to exploit and study with greater precision soybean resistance against P. sojae.     

基于SSR标记分析大豆疫霉根腐病抗源的遗传多样性

丰富的遗传多样性可为大豆育种提供宽阔的遗传基础,本研究基于35对SSR标记,对60份东北地区大豆疫霉根腐病抗性品种进行了遗传多样性分析,共检测到189个等位基因,平均每个位点等位变异数5.4个,多态性信息含量指数(PIC)为0.1550~0.8195,平均为0.6636;遗传相似系数的变异范围为0.31~0.74。利用5对高多态性SSR引物构建了60份抗性材料的指纹图谱,这5对SSR引物构建的指纹图谱可以将60份疫霉根腐病抗性材料逐一区分开。采用NTSYS2.10基于遗传距离的聚类分析,将60份抗性材料分为7个类群,其中78.33%的抗性品种(系)的遗传相似系数在0.45~0.74间,表明遗传差异相对较窄,品种间遗传多样性水平较低。聚类分析与群体遗传结构分析结果有部分重合,均反映出不同地区的抗性材料间存在一定的渗透和交流。     

用SSR标记分析抗疫霉根腐病大豆品种(系)的遗传多样性

利用50对SSR引物对抗疫霉根腐病大豆品种(系)进行遗传多样性分析。在166份品种(系)中,50个SSR座位共产生265个等位变异,平均每个座位5.3个。采用NTSYS-pc2.10计算品种(系)间遗传相似系数,平均相似系数为0.3124,表明抗疫霉根腐病大豆品种(系)间的遗传差异较大。用UPMGA进行聚类分析,166个品种(系)在相似系数为0.33时被聚为6类,地理来源相同的品种(系)大多聚类在一起。一些具有相同或相近抗病反应型的品种(系)被聚类在同一个类群中,表明这些抗病品种(系)的遗传关系较近,应有选择地利用。W illiam s和C lark抗疫霉根腐病近等基因系构成明显不同于中国大豆的基因源,可以用于拓宽我国大豆品种的遗传基础。     

大豆疫霉根腐病抗源筛选

由大豆疫霉菌引起的大豆疫霉根腐病是大豆生产的重要病害,该病已在我国大豆主要产区发生,并在局部地区造成较大产量损失。利用抗病品种是防治大豆疫霉根腐病最有效的方法。本研究目的是筛选大豆疫霉根腐病抗源,为病害防治和抗病品种的选育提供参考。用下胚轴创伤接种方法对120个栽培大豆品种（系）进行接种,鉴定其对10个具有不同毒力大豆疫霉菌菌株的抗性。有110个品种（系）分别抗1～10个大豆疫霉菌菌株,其中以河南大豆品种（系）对疫霉菌的抗性最丰富,安徽、湖北和山西大豆品种（系）也具有抗性多样性。120个大豆品种（系）对10个大豆疫霉菌菌株共产生57个反应型,有4个抗性反应型分别与单个抗病基因的反应型一致,有7个抗性反应型与2个已知基因组合的反应型相同,其他抗性反应型为新的类型。一些大豆品种（系）中可能存在有效的抗大豆疫霉根腐病新基因。     

大豆品种早熟18抗疫霉根腐病基因的SSR分子标记

大豆品种早熟18是抗疫霉根腐病的有效抗源。本研究鉴定和分子标记早熟18的抗疫霉根腐病基因,以期为该品种的有效利用及分子辅助育种奠定基础。以感病大豆品种Williams与早熟18杂交建立分离群体。抗性遗传分析表明,早熟18对大豆疫霉菌抗性由1个显性单基因控制,该基因被定名为RpsZS18。SSR标记连锁分析表明,RpsZS18位于大豆分子遗传连锁群D1b上的SSR标记Sat_069和Sat_183之间,与这两个标记的遗传距离分别为10．0cM和8．3cM。RpsZS18是D1b连锁群上鉴定的第一个抗疫霉根腐病基因。     

Real‐time PCR Suggests that Aphanomyces euteiches is Associated with Reduced Amounts of Phytophthora medicaginis in Alfalfa that is Co‐inoculated with Both Pathogens

Aphanomyces euteiches and Phytophthora medicaginis are two pathogens of seedling and mature alfalfa (Medicago sativa L.) that are frequently found in the same field sites. In order to investigate possible interactions of these two pathogens, two greenhouse experiments were conducted on seedling alfalfa from check populations representing the phenotypic classes of dual susceptibility and dual resistance to both pathogens. Seedlings were challenged with multiple inoculum concentrations of A. euteiches and P. medicaginis. Separate real‐time PCR assays specific for A. euteiches and P. medicaginis were used to quantify the amount of each pathogen in root tissue. For both pathogens, significantly more pathogen DNA was detected in the susceptible check population Saranac than in the resistant check population WAPH‐1 in all treatment combinations. In general, co‐inoculation with both A. euteiches and P. medicaginis resulted in significantly reduced amounts of P. medicaginis DNA detected when compared with amounts detected from inoculations with P. medicaginis alone. This relationship was observed for the analysis of bulked plant samples and also for individual plants. Co‐infestation by both pathogens did not reduce the quantity of A. euteiches detected. Possible mechanisms responsible for the inhibition of accumulation of P. medicaginis by A. euteiches are discussed.     

Genome-Wide Association Study of Partial Resistance to P. sojae in Wild Soybeans from Heilongjiang Province,China

Phytophthora root rot (PRR) is a destructive disease of soybeans (Glycine max (L.) Merr) caused by Phytophthora sojae (P. sojae). The most effective way to prevent the disease is growing resistant or tolerant varieties. Partial resistance provides a more durable resistance against the pathogen compared to complete resistance. Wild soybean (Glycine soja Sieb. & Zucc.) seems to be an extraordinarily important gene pool for soybean improvement due to its high level of genetic variation. In this study, 242 wild soybean germplasms originating from different regions of Heilongjiang province were used to identify resistance genes to P. sojae race 1 using a genome-wide association study (GWAS). A total of nine significant SNPs were detected, repeatedly associated with P. sojae resistance and located on chromosomes 1, 10, 12, 15, 17, 19 and 20. Among them, seven favorable allelic variations associated with P. sojae resistance were evaluated by a t-test. Eight candidate genes were predicted to explore the mechanistic hypotheses of partial resistance, including Glysoja.19G051583, which encodes an LRR receptor-like serine/threonine protein kinase protein, Glysoja.19G051581, which encodes a receptor-like cytosolic serine/threonine protein kinase protein. These findings will provide additional insights into the genetic architecture of P. sojae resistance in a large sample of wild soybeans and P. sojae-resistant breeding through marker-assisted selection.     

Phytophthora cryptogea root rot of tomato in rock woo] nutrient culture. II. Effect of root zone temperature on infection, sporulation and symptom development

The effect of root-zone temperature on Phytophthora cryptogea root rot was studied in tomato cv. Counter grown under winter and summer conditions in rockwool culture. A nutrient temperature of 25°C resulted in increased root initiation and growth, higher in winter-grown than in summer-grown plants. Rhizosphere zoospore populations were greatly reduced at 25°C and above. Growth of P. cryptogea in vitro was optimal between 20°C and 25°C and completely suppressed at 30°C. Encystment was enhanced by increased temperatures above 20°C. Zoospore release in vitro occurred in cultures maintained at constant temperatures in the absence of the normal chilling stimulus. Optimal release was at 10°C; no zoospores were released at 30°C. Inoculated, winter-grown tomato plants maintained at 15°C developed acute aerial symptoms and died after 21 days. Comparable plants grown at a root-zone temperature of 25°C remained symptomless for the 3-months duration of the experiment. Summer-grown infected plants at the higher root temperature wilted but did not die. Enhanced temperature was ineffective as a curative treatment in summer-grown plants with established infection. Aerial symptoms of Phytophthora infection are seen as a function of the net amount of available healthy root. With high root zone temperatures this is determined by new root production and decreased inoculum and infection.     

A CRISPR/Cas9-mediated in situ complementation method for Phytophthora sojae mutants

Phytophthora sojae is an important model species for oomycete functional genomics research. Recently, a CRISPR/Cas9-mediated genome-editing technology has been successfully established in P. sojae, which has been rapidly and widely applied in oomycete research. However, there is an emerging consensus in the biological community that a complete functional gene research system is needed such as developed in the investigations in functional complementation carried out in this study. We report the development of an in situ complementation method for accurate restoration of the mutated gene. We targeted a regulatory B-subunit of protein phosphatase 2A (PsPP2Ab1) to verify this knockout and subsequent complementation system. We found that the deletion of PsPP2Ab1 in P. sojae leads to severe defects in vegetative hyphal growth, soybean infection, and loss of the ability to produce sporangia. Subsequently, the reintroduction of PsPP2Ab1 into the knockout mutant remedied all of the deficiencies. This study demonstrates the successful implementation of an in situ complementation system by CRISPR/Cas9, which will greatly accelerate functional genomics research of oomycetes in the post-genomic era.     

Phytophthora cryptogea root rot of tomato in rockwool nutrient culture:

The effect of root temperature on growth and yield of rockwool-grown tomato plants infected with Phytophthora cryptogea was investigated. Measurements of shoot and root growth were taken at high (25^oC) and low (15^oC) root temperatures during the generative phase of growth. The growth of roots of healthy and P. cryptogea-infected tomato plants in rockwool blocks was higher in plants grown with roots at 25^oC than at 15^oC after 60 days and a similar effect was found in slabs after 98 days. Under sub-optimal conditions for growth the disease became severe when root temperatures were low. Growth of roots was greatest when roots were maintained at a high temperature in combination with an ambient air temperature of c. 15^oC and the response was greater in cv. Counter than cvs Calypso and Marathon. Water-soluble carbohydrates of roots were higher in those produced in blocks than slabs and were reduced by infection compared to healthy plants with roots at 15^oC and 25^oC. Reduced transpiration rates were found 17 days after inoculation in symptomless plants grown at a root temperature of 25^oC. Infection, regardless of the temperature of the roots or cultivar, led to reduced stem growth. The plants grown at 25^oC were taller than those with a root temperature of 15^oC. After 9 wk of harvest, the cumulative fruit yields in infected cvs Counter and Calypso grown at 25^oC were comparable to that in healthy plants grown at either temperature and cumulative fruit numbers followed a similar pattern. High root temperatures led to delayed fruit ripening between weeks 3–10 and a larger number of unripe fruit. The weight of unripe fruit from infected plants grown at 25^oC at the terminal harvest was higher than from healthy plants with roots maintained at 15^oC.     

Phytophthora cryptogea root rot of tomato in rockwool nutrient culture. I. Analysis of root infection

The spatial and temporal colonisation of tomato plants by Phytophthora cryptogea was studied in rockwool nutrient culture. Root growth and the distribution and progress of infection were measured on dissected root fragments obtained from a detailed destructive sampling of the substrate. Dissected fragments of root or roots and fibres were plated on BNPRA agar, a selective Phytophthora medium. Roots colonised all parts of the rockwool substrate 60 days after planting with the exception of surface marginal and central areas of the slab which had a lower solution content. Most root biomass occurred in and immediately beneath the original growing block. The distribution of P. cryptogea closely followed the pattern of root colonisation. An alternative, novel method for root analysis involved the dissolution of the mineral fibre and its formate bonding resin by digestion in 1 m H₃PO₄ for 45 min. Comparative recoveries of P. cryptogea from plated fragments of dissected root and fibre or comminuted samples of acid-released or dissected root showed that the acid treatment initially reduced the number of Phytophthora colonies in block and slab roots by 67% and 61% respectively. After 28 days, colony recovery from acid-released roots in the rockwool slab increased and was between 4% and 13% lower than from other plating methods. Since 1 m H₃PO₄ was lethal to zoospores and surface sporangia, the colonies recovered were interpreted as originating exclusively from root lesions. Root fresh weight of healthy and inoculated plants declined during the initial period of fruit formation. P. cryptogea infection led to a progressive reduction in root weight in the growing block and main slab and 28 days after inoculation, was approximately 50% of the controls. The acid digestion of rockwool fibre is proposed as a new approach to the problem of root and pathogen analysis in this substrate.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.