海南芒果蒂腐病菌对吡唑醚菌酯的抗药性测定

从海南芒果主产区采集分离蒂腐病菌Botryodiplodia theobromae。首先采用区分剂量法检测供试菌株对吡唑醚菌酯的抗药性,然后再采用生长速率法测定吡唑醚菌酯对蒂腐病菌的EC_(50)。结果表明:95株芒果蒂腐病菌中有56株在含10mg/L吡唑醚菌酯的PDA培养基中菌落直径大于空白对照的40%,抗性频率为58.95%,来自不同产区的芒果蒂腐病菌对吡唑醚菌酯的抗性频率差异不大。吡唑醚菌酯对供试菌株的EC50在3.12~1525.43mg/L之间,呈连续性正态分布,而抗性最高的菌株的EC_(50)是最敏感菌株EC50的488.92倍。敏感菌株、低抗菌株、中抗菌株及高抗菌株的平均EC_(50)分别为:5.39mg/L、45.34mg/L、144.72mg/L和514.46mg/L。海南芒果蒂腐病菌对吡唑醚菌酯杀菌剂已经产生严重的抗药性。     

The effect of the presence of R-genes for resistance to late blight (Phytophthora infestans) of potato (Solanum tuberosum) on the underlying level of field resistance

The differential genotypes R₁, R₁₀ and R₁₁, as originally defined by Black, were crossed with R-gene-free cultivars and the progenies divided into two subpopulations comprising those which had inherited the R-gene and those which had not. The underlying level of field resistance of the two groups was compared in a field trial in which they were inoculated with an isolate that could overcome the relevant R-genes. The R-gene-bearing group was significantly ( P  < 0·001) more resistant than the R-gene-free group, with mean scores over four dates in 2000 of 4·86 and 4·09, respectively, on a 1–9 scale of increasing resistance, and of 4·10 and 2·35 on one date in 2001. However, the magnitude of the effect depended on the R-gene and the year of the trial. Data from a progeny of cv. Stirling (with an R-gene and a high level of field resistance) were examined and the same effect of an R-gene found. Fewer of the R-gene-bearing group of clones were highly susceptible, and more were resistant. The most resistant clones always bore the R-gene. It is concluded that increased resistance is conferred by the defeated R-gene or linked genes for field resistance.     

几种微生物提取物诱导马铃薯抗晚疫病及机理的初步研究

 筛选能显著诱导马铃薯块茎抗晚疫病的微生物源激发子,并探讨其诱导抗病性机理。分别将31种微生物的发酵液(F-0)以及其中真菌和放线菌的胞内提取物(F-1)、胞壁提取物(F-2)单独或复配后诱导处理马铃薯块茎切片,对其中诱导抗病效果显著的激发子进一步分析其中的有效诱抗物质及诱导处理后的生理变化。获得了9种较好的激发子,诱抗效果均达50%以上,其中放线菌A5295发酵液型激发子诱抗效果最高,达63.97%,且单一激发子复配后诱抗活性更高,其中MK与A5295的复配型激发子诱抗效果最好,达66.67%。MK和A32910b发酵液中有效诱抗物质均为饱和硫酸铵沉淀(A组分)、以及硫酸铵沉淀后再用乙醇沉淀(B组分)所得的物质。诱导处理后块茎中POD、PAL、PPO活性均明显高于对照,且在抗性表现出之前迅速增加达到峰值。此外,块茎中可溶性蛋白含量比对照增加41.53%,并有一些新蛋白产生。结果表明复配激发子比单一激发子诱抗活性更高,激发子中有效诱抗物质均为A和B组分,诱导的抗病性可能与块茎中POD、PAL、PPO活性增高,可溶性蛋白含量增加以及新蛋白质的产生有关。     

磷酸二氢钾影响马铃薯对晚疫病抗性的初步研究

本研究以马铃薯品种‘丽薯6号’为试材,对叶面喷施不同浓度磷酸二氢钾4 d后的植株接种晚疫病菌,测定晚疫病发生的严重度,并测定磷酸二氢钾和晚疫病菌处理后14 d内马铃薯植株的PAL、SOD、POD、PPO、Chi、GLU活性及SP含量。结果表明,在一定浓度范围内,磷酸二氢钾可减轻马铃薯晚疫病的发生,随着施用浓度的增加,晚疫病发病逐渐减轻。其中磷酸二氢钾质量浓度为0.6%时,晚疫病发病最轻,接种晚疫病菌后8 d防效达35.64%,12 d时仍超过30%,但浓度超过0.6%,晚疫病发生加重。同时,喷施磷酸二氢钾可不同程度地提升健康和接种晚疫病菌的马铃薯植株体内6种防御酶活性及可溶性蛋白含量。因此,磷酸二氢钾在一定时间段内可诱导马铃薯对晚疫病的抗性,减轻晚疫病的发生。     

Molecular and biochemical characterization of boscalid resistance in laboratory mutants of Sclerotinia sclerotiorum

The plant‐pathogenic fungus Sclerotinia sclerotiorum has a broad host range and a worldwide distribution. Boscalid, an inhibitor of succinate dehydrogenase in the electron transport chain of fungi, is highly effective in controlling sclerotinia stem rot caused by S. sclerotiorum. The current study characterized the S. sclerotiorum boscalid‐resistant (BR) mutants obtained by fungicide induction. Among the bioactive fungicides against S. sclerotiorum, cross‐resistance was not detected between boscalid and dimethachlon, fluazinam or carbendazim; positive cross‐resistance was detected between boscalid and carboxin; and negative cross‐resistance was detected between boscalid and kresoxim‐methyl. Compared to their parental isolates, BR mutants had slower radial growth, no ability to produce sclerotia, lower virulence and oxalic acid content but higher mycelial respiration and succinate dehydrogenase (SDH) activity. Moreover, BR mutants had decreased sensitivity to salicylhydroxamic acid (SHAM) but not to oxidative stress. All the results indicated that the risk of resistance to boscalid in S. sclerotiorum is low to moderate. DNA sequence analysis showed that all of the BR mutants had the same point mutation A11V (GCA to GTA) in the iron sulphur protein subunit (SDHB). Interestingly, expression of the cytochrome b (cytb) gene was reduced to different degrees in the BR mutants, and this might be correlated with the negative cross‐resistance between boscalid and kresoxim‐methyl. Such information is vital in the design of resistance management strategies.     

Influence of rate of soil fertilization on alternaria leaf blight (Alternaria dauci) in carrots

The possibility of suppressingAlternaria dauci (Kühn) Groves & Skolko, the causal agent of Alternaria leaf blight in carrot, by excess application of fertilizer was examined in greenhouse and field experiments. Reducing the rate of fertilization by one half from the optimal rate (100 ppm N, 19 ppm P and 74 ppm K) resulted in a 23–30% increase in the severity of Alternaria leaf blight. However, doubling the rate of fertilization resulted in only a 10–15% decrease in disease severity. Inoculating with different concentrations ofA. dauci spores (10³ or 10⁴ spores/ml) did not alter the response of the plants to the fertilization rate, although significantly higher disease severity was observed in plants inoculated with the higher spore concentration. These results were corroborated in the field, where neither disease severity nor harvested yield was significantly affected by tripling the amount of soil fertilization. Application of foliar fungicides, on the other hand, had substantial effects on both disease and yield. Therefore, it was concluded that carrot crops should be fertilized and maintained for optimum yield, and thatA. dauci should be managed by properly timed applications of fungicides during the growing season. Contribution no. 533/99 from the Inst. of Plant Protection, Agricultural Research Organization.     

杭白菊叶枯病病原菌鉴定及其对多菌灵的抗性机制

为明确浙江省桐乡市发生的杭白菊叶枯病的病原菌,采用传统组织分离法对采集的病样进行病原菌分离,在测定其致病性的同时,结合形态学特征及基于核糖体内部转录间隔区(ITS)、nrDNA大亚基(LSU)和β-微管蛋白基因(TUB2)的联合系统发育分析对该病原菌进行鉴定,并测定其对多菌灵的抗性。结果表明,从杭白菊叶枯病病样中共分离到35株菌株,在回接试验中杭白菊表现出的症状与田间自然发病症状一致,证明分离到的菌株为引起杭白菊叶枯病的病原菌。该病原菌菌丝生长初期为淡黄色,后为灰白色;培养25 d后菌落上的黑色球形分生孢子器产生大量液体状的淡粉色分生孢子堆;分生孢子为单胞、无色、长椭圆形,大小(n=200)为2.8～4.9μm×1.2～3.0μm,初步判断该病原菌是茎点霉属Phoma真菌P. bellidis。基于ITS、LSU、TUB2联合系统发育分析的分子鉴定结果与形态学鉴定结果一致,确定引起该病害的病原菌为P. bellidis。该病原菌对多菌灵的抗性频率为94.3%,且全部为高水平抗性菌株,抗药性机制为其TUB2的E198A突变,即TUB2的第198位密码子从GAG突变为GCG,导致第198位氨基酸从谷氨酸(Glu)突变为丙氨酸(Ala)。     

29个马铃薯品种(品系)抗晚疫病基因组成与抗病相关性分析

由致病疫霉引起的马铃薯晚疫病是马铃薯生产中最具危害性的病害,明确现有马铃薯品种(品系)中抗病基因组成情况对于指导抗病品种合理布局及抗病育种具有重要意义。本研究利用农杆菌介导的无毒基因瞬时表达技术分析了致病疫霉8个无毒基因在29个马铃薯品种(品系)中的分布情况。结果表明,品种间抗病基因组成各异,平均每个品种含有4个抗病基因。同时,不同抗病基因在品种中的分布频率也不同,抗病基因R1(76%)、Rpi-blb1(66%)和Rpi-blb2(66%)在品种中分布较普遍;而含抗病基因R3b(45%)、R4(38%)和Rpi-vnt1(34%)的品种较少。此外,品种中所含抗性基因的数量和品种室内抗性程度呈显著正相关(r=0.915 6,P=0.029 1)。     

吉林省西瓜蔓枯病菌对吡唑醚菌酯的抗性监测及抗性风险评估

为明确吉林省西瓜蔓枯病菌Stagonosporopsis citrulli对吡唑醚菌酯的敏感基线和抗性风险,采用菌丝生长速率法测定吉林省151株西瓜蔓枯病菌菌株对吡唑醚菌酯的敏感性及吡唑醚菌酯对西瓜蔓枯病菌的最低抑制浓度,并监测375株西瓜蔓枯病菌菌株对吡唑醚菌酯的抗性水平。结果表明,供试的151株菌株对吡唑醚菌酯的敏感性频率分布呈连续的单峰曲线,接近正态分布,敏感基线为1.800 μg/mL;吡唑醚菌酯的最低抑制浓度MIC为80.000 μg/mL。抗性水平监测结果显示,375株菌株对吡唑醚菌酯的抗性频率为0。室内药剂驯化获得的抗性突变菌株T-LH3-1和T-XZC5-1在连续转接培养8代后的EC₅₀分别为16.139 μg/mL和31.782 μg/mL,抗性倍数分别为15和51,均为中抗水平,抗药性可以稳定遗传。抗性突变体及其亲本菌株生物学性状结果显示,抗性突变体生物适合度与其亲本菌株差异不显著。交互抗性结果表明,吡唑醚菌酯与醚菌酯之间存在正交互抗性,与戊唑醇、异菌脲、多菌灵和肟菌酯之间不存在交互抗性,可以交替使用以达到延缓抗药性的产生。     

Sensitivity of Phytophthora infestans to flumorph: in vitro determination of baseline sensitivity and the risk of resistance

The sensitivity of 127 Phytophthora infestans isolates to flumorph was determined in 2003 and 2004. The isolates originated from two geographical regions and showed similar levels of sensitivity in both years. Baseline sensitivities were distributed as a unimodal curve with EC₅₀ values for growth of mycelia ranging from 0·1016 to 0·3228  µ g mL⁻¹, with a mean of 0·1813 (± 0·0405) µ g mL⁻¹. There was no cross-resistance between flumorph and metalaxyl. Laboratory studies were conducted to evaluate the risk of P. infestans developing resistance to flumorph. Mutants resistant to metalaxyl or flumorph were obtained by treating mycelium of wild-type isolates with ultraviolet radiation. Metalaxyl-resistant mutants were obtained with a high frequency and exhibited resistance factor values (EC₅₀ resistant/EC₅₀ sensitive phenotypes) of more than 100, while flumorph-resistant mutants were obtained at much lower frequencies and had very small resistance factors (1·5–3·2). There was cross-resistance between flumorph and dimethomorph, but not with azoxystrobin or cymoxanil. Most flumorph-resistant mutants showed decreases in hyphal growth in vitro and in sporulation both in vitro and on detached leaf tissues. These studies suggested that the risk of resistance developing was much lower for flumorph than metalaxyl. However, as P. infestans is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Resistance status of cultivated potatoes to early blight (Alternaria solani) in Denmark

Field experiments were conducted in 2015 and 2016 to classify the resistance level to early blight of the main potato cultivars in Denmark. A total of 38 cultivars were used. The experiment was designed as a randomized complete block design (RCBD) with four replicates. The potatoes were inoculated by spreading barley (Hordeum vulgare) kernels infested with Alternaria solani and A. alternata. Disease assessment was done at weekly intervals starting from the onset of the first symptoms. The disease progress curves (DPC) of early blight on the cultivars were compared to that of Bintje. A multivariate analysis was performed with the apparent infection rate, the relative area under the disease progress curve, the time to reach 50% severity, the daily defoliation, the disease severity in the middle of the epidemic and the duration of the epidemic to classify the resistance level of the cultivars. The analysis of the DPC of the cultivars showed that none of the cultivars was completely resistant to early blight. However, the epidemic development differed between the different cultivars. The resistance levels of the cultivars were thus classified into classes I, II, III and IV interpreted as ‘very susceptible’, ‘susceptible’, ‘moderately slow blighting’ and ‘slow blighting’, respectively. All the late maturity cultivars were classified as class IV, and the earlier maturing cultivars were either class I or class II.     

Incidence,pathogenicity and diversity of Alternaria spp. associated with alternaria leaf spot of canola (Brassica napus) in Australia

Studies were undertaken to determine Alternaria spp. associated with leaf spot symptoms on canola (Brassica napus) in two cropping seasons (2015, 2016) across southern Australia. Major allergen Alt a1 and plasma membrane ATPase genes were used to identify Alternaria spp. In 2015, 112 isolates of seven Alternaria spp. were obtained, with A. metachromatica predominating. In 2016, 251 isolates of 12 Alternaria spp. were obtained, with A. infectoria predominating. Alternaria spp. isolates were morphologically and phylogenetically identified and studies to determine their pathogenicity on both B. napus (cv. Thunder TT) and B. juncea (cv. Dune) confirmed 10 species (A. alternata, A. arborescens, A. brassicae, A. ethzedia, A. hordeicola, A. infectoria, A. japonica, A. malvae, A. metachromatica and A. tenuissima) as pathogenic on both Brassica species. Alternaria ethzedia, A. hordeicola and A. malvae were recorded for the first time in Australia on any host and the record of A. arborescens was the first for New South Wales (NSW) and South Australia (SA). Other first records included A. infectoria on B. napus in NSW; A. japonica on B. napus in NSW and Western Australia (WA); A. metachromatica on any host in NSW, Victoria (VIC), WA and SA; and A. tenuissima on B. napus in NSW, SA and WA. It is evident that alternaria leaf spot on canola across southern Australia is not solely caused by A. brassicae, but that a range of other Alternaria spp. are also involved to varying degrees, depending upon the year and the geographic locality.     

Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar‐infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral‐gradient end point dilution method. The frequency of boscalid‐resistant isolates (>50% relative growth when using a spiral dilution gradient starting at 507 mg L^?1) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross‐resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR‐RFLP method to assess boscalid sensitivity in A. solani populations.     

Development of natural late blight epidemics in pure and mixed plots of potato cultivars with different levels of partial resistance

Late blight, caused by Phytophthora infestans , is the most severe disease of potato worldwide. Controlling late blight epidemics is difficult, and resistance of host cultivars is either not effective enough, or too easily overcome by the pathogen to be used alone. In field trials conducted for 3 years under natural epidemics, late blight severity was significantly lower in a susceptible cultivar growing in rows alternating with partially resistant cultivars (mixtures) than in unmixed plots of the susceptible cultivar alone. Partially resistant cultivars behaved similarly in unmixed and mixed plots. Mixtures of cultivars reduced disease progress rates and sometimes delayed disease onset over unmixed plots, but did so significantly only for the slowest epidemic. This suggests that reduction of area under the disease progress curve (AUDPC) in mixtures resulted from the cumulative action of minor effects. Disease distribution was focal in all plots at all dates, as shown by Morisita's index values significantly exceeding 1. Significant yield increases for the susceptible cultivar, and occasionally for the partially resistant ones, were observed in mixed-cultivar plots compared with single-cultivar plots. These results show that cultivar mixtures can significantly reduce natural, polycyclic epidemics in broadleaved plants attacked by pathogens causing rapidly expanding lesions.     

Evaluation of different methods for the characterization of carrot resistance to the alternaria leaf blight pathogen (Alternaria dauci) revealed two qualitatively different resistances

Alternaria leaf blight (ALB), caused by Alternaria dauci, is one of the most damaging foliar diseases of carrot worldwide. The aim of this study was to compare different methods for evaluating levels of carrot resistance to ALB. Three techniques were investigated by comparison with a visual disease assessment control: in vivo conidial germination, a bioassay based on a drop‐inoculation method, and in planta quantification of fungal biomass by quantitative PCR (Q‐PCR). Three carrot cultivars showing different degrees of resistance to A. dauci were used, i.e. a susceptible cultivar (Presto) and two partially resistant genotypes (Texto and Bolero), challenged with an aggressive or a very aggressive isolate of A. dauci. Both partially resistant genotypes produced a higher mean number of germ tubes per conidium (up to 3·42±0·35) than the susceptible one (1·26±0·18). The drop‐inoculation results allowed one of the partially resistant genotypes (Bolero, log₁₀(S+1) = 1·34±0·13) to be distinguished from the susceptible one (1·90±0·13). By contrast, fungal growth measured by Q‐PCR clearly differentiated the two partially resistant genotypes with log₁₀(I) values of 2·77±0·13 compared to the susceptible cultivar (3·65±0·13) at 15 days post‐inoculation. This result was strongly correlated (r² = 0·91) with the disease severity index scored at the same date. Data obtained with the different assessment methods strongly suggest that the Texto and Bolero genotypes have different genetic resistance sources.     

Cultivar reactions and the genetic basis of resistance to alternaria stem canker (Alternaria alternata f.sp. lycopersici) in tomato

The reactions of several tomato cultivars and the genetic basis of resistance to an isolate of Alternaria alternata f. sp. lycopersici were examined. Among the 105 cultivars tested only two, Regal and Toper, were susceptible, while the remainder were highly resistant. The resistant cultivar ACE 55 VF (P_;) was crossed with the susceptible cultivar Earlypak No. 7 (P₁)- When F₁, F₂, F₃, BC₁, BC₁ selfed, F₂ X P₂ and BC₁P₂ x P₁ progenies were inoculated at the two-leaf stage with a suspension of spores of the pathogen, the ratios of resistant to susceptible plants indicated that resistance was conferred by a single dominant gene.     

Integrated control of potato late blight: predicting the combined efficacy of host resistance and fungicides

Integrating cultivars that are partially resistant with reduced fungicide doses offers growers an opportunity to decrease fungicide input but still maintain disease control. To use integrated control strategies in practice requires a method to determine the combined effectiveness of particular cultivar and fungicide dose combinations. Simple models, such as additive dose models (ADM) and multiplicative survival models (MSM), have been used previously to determine the joint action of two or more pesticides. This study tests whether a model based on multiplicative survival principles can predict the joint action of fungicide doses combined with cultivars of differing partial host resistance. Data from eight field experiments on potato late blight (Phytophthora infestans) were used to test the model; the severity of foliar blight was assessed and scores used to calculate the area under the disease progress curve (AUDPC). A subset of data, derived from the most susceptible cultivar, King Edward, was used to produce dose–response curves from which parameter values were estimated, quantifying fungicide efficacy. These values, along with the untreated values for the more resistant cultivars, Cara and Sarpo Mira, were used to predict the combined efficacy of the remaining cultivar by fungicide dose combinations. Predicted efficacy was compared against observations from an independent subset of treatments from the field experiments. The analysis demonstrated that multiplicative survival principles can be applied to describe the joint efficacy of host resistance and fungicide dose combinations.