Alteration of physiological parameters of Austrocedrus chilensis by the pathogen Phytophthora austrocedrae

The effect of the pathogen Phytophthora austrocedrae on tree physiology of Austrocedrus chilensis in Patagonia was studied in a 4‐week study. In the first week, stem‐inoculated saplings showed a significant decrease in photosynthesis (A) without alteration of stomatal conductance (g_s) or stem‐specific hydraulic conductivity (Ks). From the second week on, progressive decreases in A, g_s and Ks were observed, concomitantly with development of significant stem lesions. Water use efficiency (WUE) increased in the second week and declined progressively from the third week. Hyphae and resinous materials were observed in tracheids and rays below lesions. Necrosis of parenchyma ray cells and blockage of tracheids torus were observed. Healthy xylem showed no resinous materials or tracheid blockage, but abundant starch in rays, which was absent in altered xylem. The culture filtrate (CF) of the pathogen was shown to induce changes in extracellular pH and conductivity, and increased necrosis in tissues of leaves and stem challenged with CF in vitro. Similar results were obtained in leaf tissues of the inoculated saplings in vivo. CF injection into xylem of saplings induced a decline in A and disturbance of leaf tissue integrity, without altering g_s, WUE or Ks. The decrease of A correlated with changes in tissue integrity. A possible mechanism of A. chilensis decline induced by P. austrocedrae is discussed.     

Evidence of low levels of genetic diversity for the Phytophthora austrocedrae population in Patagonia,Argentina

Phytophthora austrocedrae is a recently discovered pathogen that causes severe mortality of Austrocedrus chilensis in Patagonia. The high level of susceptibility of the host tree, together with the distribution pattern of the pathogen, have led to the hypothesis that P. austrocedrae was introduced into Argentina. The aim of this study was to assess the population structure of P. austrocedrae isolates from Argentina in order to gain an understanding of the origin and spread of the pathogen. Genetic diversity was determined based on amplified fragment length polymorphisms (AFLPs). In total, 48 isolates of P. austrocedrae were obtained from infected A. chilensis trees, representing the geographical range of the host. Four primer combinations were used for the AFLP analysis. Of the 332 scored bands, 12% were polymorphic. Gene diversity (h) ranged from 0·01 to 0·03; the Shannon index (I) ranged from 0·01 to 0·04. A high degree of genetic similarity was observed among the isolates (pairwise S values = 0·958–1; 0·993 ± 0·009, mean ± SD). A frequency histogram showed that most of the isolate pairs were identical. Principal coordinate analysis using three‐dimensional plots did not group any of the isolates based on their geographical origin. The low genetic diversity (within and between sites) and absence of population structure linked to geographic origin, together with the aggressiveness of the pathogen and the disease progression pattern, suggest that P. austrocedrae might have been introduced into Argentina.     

Pathogenicity of Aphanomyces spp. from Different Leguminous Crops in Sweden

Host range and pathogenicity of a range of Aphanomyces spp. isolates obtained from pea roots but also from a range of other field-grown leguminous crops in southern Sweden was investigated. The Aphanomyces euteiches isolates originating from pea and the few obtained isolates originating from alfalfa, green bean and yellow sweet-clover were highly pathogenic only to pea. The A. euteiches isolated from common vetch differed from these isolates by being weakly pathogenic to pea and other legumes, but highly pathogenic to common vetch. Vetch isolates also formed a well-defined separate cluster based on principal component analysis of pathogenicity pattern on tested crops. Oospores of A. euteiches were observed in root tissue of pea as well as common vetch, alfalfa, green bean, broad bean, red clover and yellow sweet-clover in the greenhouse pathogenicity tests. An Aphanomyces sp. that morphologically differed from A. euteiches, was frequently isolated from several leguminous plants, but was non-pathogenic to all tested crops in the pathogenicity tests. In isozyme analysis the banding pattern of these isolates resembled the pattern of A. cladogamus. Another, different and so far unidentified Aphanomyces sp. from roots of green bean and broad bean, was also non-pathogenic to the tested legume species. Based on the isolates tested, the results obtained suggest that the population of Aphanomyces spp. infecting legume roots in Sweden consists of a pea-specific and a vetch-specific group of A. euteiches. Two other groups comprised (i) Aphanomyces sp. isolates that resembled A. cladogamus, and (ii) isolates, which resembled neither A. euteiches nor A. cladogamus. In addition, the host range of Swedish A. euteiches isolates was not as broad as reported for A. euteiches isolates from other countries.     

Coinfection of soybean plants with Phytophthora sojae and soybean cyst nematode does not alter the efficacy of resistance genes

The soybean cyst nematode (SCN) Heterodera glycines and the oomycete Phytophthora sojae are among the most damaging pathogens of soybean worldwide. Resistant cultivars are commonly used to manage these diseases. As it is known that the presence of SCN can facilitate the development of other pathogens, it is important to verify if there is a synergistic activity between SCN and P. sojae. The purpose of this study was to evaluate a possible interaction on susceptible and resistant soybean lines. The plants were inoculated with one or both organisms at different stages (5 or 10 days old). Two levels of SCN inoculum (2,000 and 10,000 eggs/plant) and different timing between SCN and P. sojae inoculation (2, 5, or 8 days) were compared. The results on 5-day-old plants showed that SCN did not influence P. sojae development. The resistant cultivar to P. sojae remained effective (0% mortality) and susceptible cultivars exhibited high mortality (100%) in the presence or absence of SCN. Experiments on 10-day-old plants showed that SCN resistance was not affected by the presence of P. sojae. SCN inoculum density and timing of P. sojae infection did not affect the virulence of these pathogens and the efficacy of resistance genes. However, the number of SCN cysts was decreased by more than 50% (p < .001) when P. sojae was coinfesting the susceptible cultivar. This suggests that P. sojae might indirectly influence SCN development by reducing the root mass. This study confirmed that resistant cultivars remain a valid option for the management of P. sojae and SCN.     

Detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. palmivora</Emphasis> in citrus roots using PCR-RFLP in comparison with other methods

Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensitivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000× more sensitive than a single-step PCR with primers ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-specific primers in all samples inoculated with this species despite the absence of species-specific patterns in RFLP. In contrast, the incidence of detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the target species is established prior to the interpretation of data.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Specific Behaviour of French Aphanomyces Euteiches Drechs. Populations For Virulence and Aggressiveness on Pea, Related to Isolates from Europe, America and New Zealand

The pathogenic variability of Aphanomyces euteiches on pea was investigated using a collection of 88 pea-infecting isolates from France and 21 isolates from Denmark, Sweden, Norway, USA, Canada and New Zealand. Aggressiveness and virulence were assessed by scoring the root symptoms on a differential set of six pea genotypes. Eleven virulence types were characterised. The virulence type I, previously described as virulent on the whole set, was predominant and included the most aggressive isolates of all geographical origins. The other types were much less prevalent, existing as one to five isolates. Three virulence types (III, IV and V) contained no French isolates. The type III, avirulent on MN313, was composed of American isolates only, and resembled the major group recently described in the USA. A wide range of aggressiveness was found within the virulence type I, and the French isolates appeared globally more aggressive than the foreign isolates. These findings indicate that isolates from the virulence type I should be used as references in breeding programs, and that pea lines PI180693 and 552 may be the most interesting resistance sources to date, despite their only partial resistance.     

Analysis of population structure of Rosellinia necatrix on Cyperus esculentus by mycelial compatibility and inter-simple sequence repeats (ISSR)

Genetic diversity was studied in a population of 24 isolates of Rosellinia necatrix obtained from Cyperus esculentus and 16 from other hosts by means of mycelial compatibility groups (MCGs) and inter-simple sequence repeat (ISSR) techniques. All isolates obtained from C. esculentus belonged to a unique MCG, irrespective of their geographical origin or year of isolation, and were incompatible with the isolates from other hosts. ISSR analysis gave results which were in agreement with the MCG determination; selected ISSR primers grouped a subset of eight representative isolates from C. esculentus in a unique cluster. The homogeneity found within the population of R. necatrix from C. esculentus in Valencia province could suggest that the pathogen was introduced recently and has spread further via infected tubers inadvertently used as propagating material, as well as by cull tubers swept away by irrigation water.     

Characterization of <Emphasis Type="Italic">Phytophthora clandestina</Emphasis> races on <Emphasis Type="Italic">Trifolium subterraneum</Emphasis> in Western Australia

Phytophthora clandestina is a causal agent of root rot disease of subterranean clover in Western Australia (W.A). As a significant number of isolates of P. clandestina from W.A. could not previously be designated using existing differentials, a comprehensive set of subterranean clover (Trifolium subterraneum) cultivars was used as differentials to delineate a broader range of races of the pathogen. One hundred and one isolates of the pathogen collected from W.A. were screened on nine subterranean clover cultivars, of which seven were found to be useful as host differentials. A total of 10 races (in contrast to the five recognized previously) were defined and differentiated using octal nomenclature, presenting a clearer picture of the racial distribution of P. clandestina among W.A. isolates. Differences were found in the race populations between Australian states and are therefore important to the selection/breeding of cultivars for specific regions of Australia to counter the predominant race populations and for enforcing quarantine measures in relation to seed movements within and outside Australia. The octal nomenclature used provides a sound basis for follow-up studies and future race designations. Races 173 and 177 in this study were widely distributed and were the most common races in W.A., and together constitute 80% of the isolates characterized. While six of the seven host differentials were resistant to isolates belonging to race 001 and all were resistant to 000, it is of concern that only one differential was resistant to 157 and 173 and that none of the host differentials were resistant to 177. Our approach to P. clandestina race delineation is clearly conservative and is different from previous studies. The octal nomenclature we applied in this study is not only scientifically sound but also will facilitate rapid recognition and characterization of the races.     

Pathogenicity and fungicide sensitivity of Pythium and Phytopythium spp. associated with soybean in the Huang-Huai region of China

Pythium and Phytopythium spp. cause seed decay, damping off, and root rot in soybean, wheat, and many other crops. However, their diversity and importance as pathogens, particularly in different crop rotation systems, are largely unknown. A survey was conducted in the Huang-Huai region, one of the main areas of soybean–wheat rotation farming in China. In 2016–2018, we collected 300 soybean seedlings and 150 field soil samples from several representative locations, and identified 26 Pythium and 6 Phytopythium spp. from 212 isolates, based on internal transcribed spacer 2 (ITS2) and cytochrome oxidase subunit 1 sequences. The pathogenicity of these isolates was evaluated by growing soybean and wheat seeds in dishes and pots containing oomycete cultures. We found that 12 Pythium spp. (but no Phytopythium spp.) showed high pathogenicity on soybean and/or wheat, and nine of them (75%) were highly pathogenic on both crops. Among the nine species, Pythium spinosum, Pythium ultimum, Pythium species 1 (tentatively designated as ‘Candidatus Pythium huanghuaiense’), Pythium aphanidermatum, and Pythium myriotylum were highly abundant among all isolates (15%, 10%, 9%, 8%, and 5%, respectively). Nine species were selected for testing of sensitivity to the fungicides metalaxyl and mefenoxam. The EC₅₀ values were all less than 10 μg/ml, indicating little resistance. Minimum inhibitory concentration values indicated isolates were about twice as sensitive to mefenoxam as to metalaxyl. These results provide a systematic understanding of Pythium and Phytopythium species associated with soybean in the Huang-Huai region, which is important for disease management and breeding programmes.     

Tracking Phytophthora infestans with SSR markers within and between seasons – a field study in Sweden

The dynamics of a late blight epidemic and sexual reproduction in Phytophthora infestans were studied in an experimental field in mid‐Sweden. The field was inoculated with six isolates of P. infestans taken from another potato field where sexual reproduction of the pathogen was suspected. Three weeks after inoculation single‐lesion leaflets were sampled and the resulting isolates characterized using microsatellites (SSRs) and mating type as markers. Among the 151 isolates analysed, the inoculum genotypes constituted more than 80% of the genotypes found, with three other genotypes making up the remainder. The following year, P. infestans obtained from soil samples taken from this field were analysed, and six novel genotypes were identified. Genotypes from the previous summer’s population were not detected. Analysis of the genotypes recovered was consistent with them being recombinants, with the previous summer’s population acting as parents. These findings are consistent with the hypothesis that oospores produced during a summer epidemic in Sweden can overwinter and cause infection the next year.     

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central‐eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10–30 cm, and 10–20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species‐specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open‐root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma‐complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0·004 to 1904·8 pg μL^?1. In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black‐foot pathogens.     

Pathogenicity ofPythium species on cucumber in peat-sand,rockwool and hydroponics

Pythium spp. that cause damping-off of seedlings also can cause root rot of older plants and lead to yield reductions. This can be especially severe in soilless cultures where the fungus can spread easily with the nutrient solution. 39Pythium isolates obtained from discolored roots were assayed for their ability to cause damping-off on cucumber seedlings in sand-peat and for their pathogenicity in soilless culture of cucumber in rockwool or hydroponic solution. Isolates ofPythium aphanidermatum, P. irregulare, P. sylvaticum andP. ultimum were highly pathogenic in sand-peat, but onlyP. aphanidermatum strains were pathogenic in soilless conditions and led to root decay, plant death in rockwool culture and growth reduction in hydroponic culture. One strain ofP. aphanidermatum significantly reduced the yield of cucumber grown in rockwool under conditions similar to those of commercial cultures.     

Pathogenicity variation and DNA polymorphism of Bipolaris sorokiniana infecting winter wheat in the Huanghuai floodplain of China

Wheat root rot, caused by Bipolaris sorokiniana, has led to severe losses of wheat products worldwide. To evaluate the pathogenicity and genetic variation of B. sorokiniana, diseased wheat samples were collected from 97 locations in the Huanghuai floodplain of China in 2014 and 2015 for analysis. A total of 673 isolates were obtained, 262 of which were identified as B. sorokiniana. Pathogenicity analysis of the isolates revealed variation in pathogenicity, which was not directly correlated with geographic region. Large variations in pathogenicity were also found within geographic groups. To determine the genetic structure of the populations, PCR was performed with universal rice primers (URP). Cluster analysis based on amplification patterns showed that the classified groups were correlated with geographical regions. Thus, analysis of the genetic diversity of the population indicated a negative correlation with geographic origin, that is, the greater the distance between sites, the lower the genetic variation similarity coefficient. Identification of wheat germplasm resistance showed that resistant cultivars accounted for a low percentage, while susceptible and highly susceptible cultivars were in the majority. Overall, these results are meaningful for developing strategies to prevent and control wheat root rot.     

海洋细菌解淀粉芽胞杆菌SH-27在大豆体内的定殖动态及促生防病作用

为明确海洋细菌SH-27在大豆体内的定殖动态及其促生长作用和对大豆疫病的防治效果,本研究采用抗利福平标记法和平板对峙生长法,筛选对利福平标记稳定且对大豆疫霉菌具有较好抑菌作用的标记菌株SH-27^Rif,培养10代后的标记菌株SH-27^Rif能够保持稳定,对大豆疫霉抑制率为56.92%。分别采用灌根和涂叶法研究其在大豆体内的定殖动态,灌根与涂叶法均可使标记菌株SH-27^Rif在大豆体内定殖,时间达31 d以上;灌根处理定殖量呈先升后降趋势,定殖量根 > 茎 > 叶,处理后第21 d根部定殖量达到最大（6.6×10⁵ cfu/g）;涂叶处理第1 d大豆叶片定殖量达到最大（6.3×10⁵ cfu/g）,随后呈迅速下降趋势;定殖量叶>茎,根部未检测到标记菌株SH-27^Rif。盆栽促生试验结果表明,菌株SH-27发酵液灌根处理第15 d,处理组株高、根长、茎粗、鲜重、干重、叶绿素含量、根系活力等指标均显著高于对照组。盆栽防病试验结果表明：菌株SH-27发酵液灌根处理能显著降低大豆疫病的病情指数,对大豆疫病3、5、7和9 d的防效分别为83.44%、66.34%、57.18%和52.85%。以上研究结果表明海洋细菌SH-27是防治大豆疫病的潜在生防菌株,具有良好的开发和应用价值。     

甘薯根腐病抗性在不同环境条件下的表现及遗传趋势

结果表明,甘薯根腐病发病轻重与环境条件有很大关系,表现为干旱少雨的年份发病较重,降雨量较多的年份发病较轻;土壤瘠薄的发病较重,肥力条件较好的则发病较轻;通常情况下,年份间品种的抗性表现较为一致,但遇特殊气候则年份间品种的抗性有一定的差异。对1150份甘薯品种资源及育种材料的根腐病抗性鉴定结果表明,高抗型材料占14.6％,抗病型占15.7％,感病型占26.0％,高感型占43.7％。对754份材料及亲本的抗性分析表明,不同的抗性组合后代中均可分离出高抗至高感类型的材料,杂交后代的抗性强弱随双亲抗性水平的提高而提高。中国自1970年以来采用品种间杂交和种间杂交育种技术,先后育成了一批高产、优质的高抗型优良品种。     

党参和黄芪水提液对当归根腐病病原菌的抑制作用研究

当归根腐病是当归生产中的重要病害之一,分离纯化后鉴定为尖孢镰刀菌(Fusarium.oxysporum)为了明确中草药党参和黄芪提取液对当归根腐病的抑制作用,分别以两种中药水浸液对小当归根腐病的抑制作用进行测定。结果表明:不同浓度的中草药党参和黄芪提取液对当归根腐病菌丝生长和孢子萌发都有抑制作用,在浓度为0.50 g/mL对当归根腐病菌丝生长的抑制率为25.23%和53.48%,对其孢子萌发抑制率为51.57%和74.56%,且所有处理发生根腐病均比对照低,差异达显著水平。     

Genetic diversity of Phytophthora pluvialis,a pathogen of conifers,in New Zealand and the west coast of the United States of America

Phytophthora pluvialis is the causal agent of red needle cast on Pinus radiata in New Zealand. It was first isolated in 2008 but had previously been recovered from tanoak (Notholithocarpus densiflorus) and Douglas‐fir (Pseudotsuga menziesii) trees in Oregon, USA in 2002. Phytophthora pluvialis was subsequently described as a new species in 2013 and classified as a clade III Phytophthora species. The aim of this study was to gain a better understanding of the genetic diversity, population structure and origin of this pathogen. A total of 360 P. pluvialis isolates were collected from the USA and New Zealand. The genome sequences of two P. pluvialis isolates were used to identify 27 single nucleotide polymorphism (SNP) markers that were then used to genotype the two populations. The genotypic data showed that the USA population of P. pluvialis had twice the genetic diversity and a greater number of multilocus genotypes (MLGs) compared to the New Zealand population, with 126 and 24 MLGs, respectively. The majority of the subpopulations within the USA and New Zealand showed linkage disequilibrium. All subpopulations had a negative fixation index, indicating that clonal reproduction is prevalent in both countries. A minimum spanning network (MSN) showed two unique clusters of isolates in the New Zealand population, suggesting two potential introductions of P. pluvialis into New Zealand from the USA. There was no significant structure within the New Zealand or USA populations. This study provides novel insight into the genetic structure of P. pluvialis in New Zealand and the USA.     

云南白背飞虱不同种群对杀虫剂的抗药性及变化趋势

为了掌握云南不同稻区白背飞虱Sogatella furcifera(Horváth)种群对常用杀虫剂的抗药性及其发展趋势,2012-2015年,连续4年采用稻茎浸渍法在室内测定了滇西沧源、滇西南勐海和滇东师宗3个稻区田间白背飞虱对5种常用杀虫剂的抗药性。云南不同稻区田间白背飞虱对5种药剂的抗性水平总体在0.9~23.4倍,其中对毒死蜱的抗药性最高,除少数年份外,LC502 mg/L,抗性倍数10倍,抗药性达到了中等水平,年度间变化幅度较大;对噻嗪酮的抗药性呈下降趋势,滇西沧源和滇西南勐海种群对噻嗪酮的抗药性由2012年的中抗水平(11.0倍和23.4倍)下降到2015年的4.6倍和7.5倍;3个稻区种群4年对吡虫啉、吡蚜酮和噻虫嗪的抗性倍数最高达7.2、6.5和5.5倍,总体表现为无抗或低水平抗性。由于噻嗪酮和毒死蜱在云南稻区的频繁和广泛使用,田间白背飞虱已对其产生抗药性,建议限制这两种杀虫剂的使用频率或与不同作用方式的药剂轮换使用,其余药剂可以有计划地选用。