灰葡萄孢对腐霉利的抗性分子机制及快速检测技术

为明确灰葡萄孢Botrytis cinerea对腐霉利的抗性现状,于2017—2018年采用单孢分离法从浙江省5个地区的草莓大棚共分离获得200个菌株。通过区分剂量法测定了其对腐霉利的抗性,对抗药性菌株的分子机制进行了分析,并根据抗药性分子机制,建立了B. cinerea腐霉利高抗基因型的环介导等温扩增 (loop-mediated isothermal amplification, LAMP) 检测技术。结果表明:浙江省草莓B. cinerea群体对腐霉利的抗性频率高达71.5%,以低抗菌株为主。抗药性菌株的BcOS1基因上存在3种类型的突变:第Ⅰ类为BcOS1基因第365位密码子由ATC突变为AGC,导致编码的氨基酸由异亮氨酸 (Ile, I) 突变为丝氨酸 (Ser, S);第Ⅱ类为BcOS1基因第365位密码子由ATC突变为AAC,导致编码的氨基酸由异亮氨酸 (Ile, I) 突变为天冬酰胺 (Asn, N)。这两类单点突变均导致B. cinerea对腐霉利表现低或中水平抗性。第Ⅲ类包含两个连锁的突变位点,第369位和第373位密码子分别由CAG和AAC突变为CCG和AGC,导致氨基酸分别由谷氨酰胺 (Gln, Q) 和天冬酰胺 (Asn, N) 突变成脯氨酸 (Pro, P) 和丝氨酸 (Ser, S),使得B. cinerea对腐霉利表现高水平抗性。本研究所建立的LAMP检测技术可在63 ℃恒温条件下,在50 min内完成对腐霉利高抗菌株Q369P的检测,最低检测限为10 × 10?3 ng/μL,灵敏度是常规PCR的10倍。本研究结果可为腐霉利在草莓灰霉病防治上的科学使用及抗药性治理提供理论依据和技术手段。     

樱桃采后灰霉病菌对甲基硫菌灵、乙霉威和腐霉利的抗性

本文采用单孢分离法对四川汉源和山东烟台等地采集的樱桃果实进行了采后灰霉病的病原菌分离和鉴定;采用区分剂量法分别测定了菌株对苯并咪唑类杀菌剂甲基硫菌灵、乙霉威和二甲酰亚胺类杀菌剂腐霉利的敏感性,并进一步分析了抗药性菌株的分子机制。结果表明,分离得到的54株樱桃采后灰霉病菌均为灰葡萄孢Botrytis cinerea,对甲基硫菌灵的总抗性频率高达79.6%,其中甲基硫菌灵抗性-乙霉威敏感 (BEN R1) 菌株频率为 25.9%;甲基硫菌灵-乙霉威双重抗性菌株 (BEN R2) 频率为53.7%;检测到腐霉利抗性菌株 (DCF R) 9 株,频率为16.7%。甲基硫菌灵抗性菌株在β-tubulin基因上的突变共有2种类型: BEN R1抗性菌株中,第198位密码子发生点突变 (GAG→GCG),编码氨基酸由Glu (E)突变成缬氨酸Ala (A);在BEN R2抗性菌株中,第198位密码子发生点突变 (GAG→GTG),编码氨基酸由Glu (E)突变成缬氨酸Val (V)。DCF R菌株在BcOS1的第365位密码子由ATC突变成AAC或AGC,导致编码的氨基酸由异亮氨酸Ile (I)突变成天冬酰胺Asn (N)或丝氨酸Ser (S)。本研究表明樱桃采后灰霉病菌对甲基硫菌灵和腐霉利存在不同程度抗性,应在加强抗药性监测的同时与其他类型杀菌剂交替使用,延缓抗药性发展。     

黑龙江和云南葡萄产区灰霉病菌对多菌灵和异菌脲的抗性检测

为明确黑龙江省和云南省葡萄灰霉病菌对多菌灵和异菌脲的抗性频率, 从两省分别采集?分离到葡萄灰霉病菌 Botrytis cinerea 39株和38株, 利用菌丝生长速率法测定其对多菌灵和异菌脲的敏感性, 并分别检测上述菌株两种药剂靶标基因 β-tubulin 和 Bos1 的突变情况?结果表明, 黑龙江省葡萄灰霉病菌对多菌灵和异菌脲的抗性频率以及对两种杀菌剂的多药抗性频率分别为100.0%?51.3%和51.3%; 云南省葡萄灰霉病菌对多菌灵和异菌脲的抗性频率以及对两种杀菌剂的多药抗性频率均为97.4%?两地区的多菌灵抗性菌株的 β-tubulin 基因上均发生了E198A或E198V突变?异菌脲抗性菌株的 Bos1 基因存在I365N?I365S?Q369P+N373和Q369P等4种突变类型?研究结果表明, 在上述两个地区不宜单独使用多菌灵和异菌脲防治葡萄灰霉病, 应使用其他作用机制不同且抑菌效果好的杀菌剂?     

设施蔬菜灰霉病菌对不同类型杀菌剂的抗性检测

为了明确设施蔬菜灰霉病菌Botryotinia fuckeliana的抗药性现状,采用菌丝生长速率法检测了20052006年采自浙江、江苏、山东和辽宁4省的144个菌株对6种常用防治药剂的敏感性。结果表明,灰霉病菌对百菌清已经产生了低水平的抗性,频率为5.56%,其对多菌灵的抗性非常严重,总的抗性频率为43.05%,高抗(HR)频率为27.08%;乙霉威的EC50值在0.137728.9μg/mL之间,平均为40.06μg/mL。其中多菌灵-乙霉威双抗频率为36.11%,且首次检测到了两种新的双抗类型。二甲酰亚胺类杀菌剂在生产上已经应用近20年,但灰霉病菌对异菌脲和腐霉利只有频率为20%左右的低水平抗性,没有检测到高抗菌株;苯胺嘧啶类杀菌剂嘧霉胺虽然只应用几年时间,但已经产生了抗性,其抗性菌株频率为4.16%。研究表明,设施蔬菜灰霉病菌对常用的6种防治药剂均产生了不同程度的抗性。     

上海市草莓灰霉病菌对氟吡菌酰胺敏感性检测及 抗性分子机制

为明确上海地区草莓灰霉病菌 Botrytis cinerea 对琥珀酸脱氢酶抑制剂类(succinate dehydrogenase inhibitors, SDHIs)杀菌剂氟吡菌酰胺的敏感性水平及抗性机制, 本研究采用菌丝生长速率法测定了2019年上海市5个草莓主产区的90个灰葡萄孢菌株对氟吡菌酰胺的抗性, 并分析了菌株的琥珀酸脱氢酶(SHD)亚基序列?结果显示:敏感性频率分布基线呈一个连续单峰曲线, 符合正态分布, 平均EC50为1.68±0.91)μg/mL, 氟吡菌酰胺对上海地区草莓灰霉病菌菌丝生长的EC50最低值为0.09 μg/mL, 最高值为75.91 μg/mL?根据新建立的抗性划分标准, 发现上海奉贤区已存在抗氟吡菌酰胺的菌系, 抗性频率72.77%, 抗性菌株的最高抗性倍数为45.18, 其他地区抗性频率较低, 浦东新区和嘉定区分别为25.00%和4.76%, 崇明?青浦区菌株的抗性频率均为0?已有抗性的菌群抗性突变位点在 Sdh B亚基的N230I位和P225F位这二个位点, 突变后分别导致低抗?中抗菌株的产生?     

上海地区草莓灰霉病菌对啶酰菌胺的抗性现状研究

为研究上海地区草莓灰霉病菌对啶酰菌胺的抗性现状,本文在上海采集了草莓灰霉病样,共分离纯化得到115株草莓灰霉病菌(Botrytis cinerea)。采用菌丝生长速率法测定了草莓灰霉病菌对啶酰菌胺的敏感性数据。结果表明,所测定的115株灰霉菌株中抗性菌株的比例占13. 9%,以中抗水平为主,共检测到16株抗性菌株,其中12株为中抗菌株,低抗和高抗菌株各检测到2株。田间试验数据显示,啶酰菌胺对草莓灰霉病3次药后防效在80%左右。综合两方面的数据,目前上海地区草莓灰霉病菌对啶酰菌胺已经产生了一定的抗性,需要引起重视,但啶酰菌胺在实际生产中用以防治草莓灰霉病仍是可行的。     

草莓灰霉病菌对嘧菌酯的抗性检测及抗性菌株的生物学特性研究

由灰葡萄孢(Botrytis cinerea Pers.)引起的草莓灰霉病是严重危害草莓的病害之一。为了解草莓灰霉病菌对杀菌剂嘧菌酯的抗性情况,本研究针对2012年从国内10省市采集和分离的134株草莓灰霉病菌菌株,利用特异性引物BcAR-F/BcAR-R扩增其cytb基因的部分序列,进行了嘧菌酯抗性的分子检测;对检测出的抗性菌株在嘧菌酯浓度分别为22.4、112和224μg·mL~(-1)的含药培养基上进行抗性验证;此外,还比较了抗性菌株与敏感菌株主要生物学性状的差异。结果表明,有54个菌株对嘧菌酯表现抗性,均属于中等抗性水平,占检测总数的40.3%,分布于北京市、湖北、江苏、河北、辽宁和四川各省;对15个抗性菌株cytb基因的序列分析表明,其第143位发生突变,由甘氨酸变为丙氨酸;对随机选取的4株抗性菌株和2株敏感菌株的适合度测定结果发现,抗性菌株和敏感菌株在最适生长温度、菌落生长速度、孢子萌发率方面没有表现出规律性差异,但抗性菌株的产孢量和致病力均显著低于敏感菌株。     

上海地区草莓灰霉病菌对啶酰菌胺的敏感性检测及抗性机制分析

为明确上海地区草莓灰霉病菌Botrytis cinerea Pers. 对主要防治药剂啶酰菌胺的敏感性水平及抗性机制,采用平皿法检测了采自上海市6个区县的195株草莓灰霉病菌株对啶酰菌胺的敏感性,并分析了其中20株不同敏感型菌株的琥珀酸脱氢酶基因序列。结果显示:啶酰菌胺对上海地区草莓灰霉病菌菌丝生长的EC₅₀最小值为0.15 μg/mL,最大值大于110 μg/mL;对孢子萌发的EC₅₀最小值为0.19 μg/mL,最大值大于50 μg/mL。上海地区草莓灰霉病菌对啶酰菌胺的抗性频率为29.74% (抗性水平大于10),高抗频率为20.51% (抗性水平大于100)。该抗性的产生与琥珀酸脱氢酶SdhB亚基发生H272R或P225F突变有关,其中H272R突变发生较为普遍。研究表明,上海地区草莓灰霉病菌对啶酰菌胺的抗性水平及抗性频率较高,主要抗性机制为病原菌琥珀酸脱氢酶SdhB亚基上的H272R突变。     

湖南省草莓灰霉病菌对4种杀菌剂的抗药性检测

为明确湖南省草莓灰霉病菌菌株的抗药性状况,2013-2015年从湖南省不同地区草莓灰霉病病果或病叶上经单孢分离获得草莓灰霉病菌454株,采用最小抑制浓度法(MIC)检测不同地区草莓灰霉病菌株对多菌灵、腐霉利、异菌脲、嘧霉胺的抗药性。结果表明:草莓灰霉病菌对多菌灵、腐霉利、异菌脲、嘧霉胺的抗性频率分别为55.73%,77.31%、3.52%和77.31%;所测菌株对4种杀菌剂的敏感性类型有Cbz~RPcm~RIpd~RPmt~R、CbzSPcm~RIpd~RPmt~R、Cbz~RPcm~RIpdSPmt~R、Cbz~SPcm~RIpdSPmt~S、Cbz~RPcm~SIpd~SPmt~R、Cbz~SPcm~RIpd~SPmt ~R等6种,其所占比例分别为33.26%、5.07%、41.41%、4.63%、3.96%、11.67%,未发现对4种杀菌剂均敏感的类型,表明湖南省草莓灰霉病菌已对多菌灵、腐霉利和嘧霉胺产生抗药性,对异菌脲的抗药性较低。     

北京地区草莓灰霉病菌的转座子及其分布频率

为了解北京地区草莓灰霉病菌的转座子类型及其分布,本研究用转座子Boty和Flipper的特异性引物对北京地区2012-2013年从12个草莓园采集和分离的60株草莓灰霉病菌进行PCR扩增。结果表明,北京地区草莓灰霉病菌群体中共存在3种转座子类型:transposa型、Boty型和Flipper型。其中,以transposa型菌株最多,占供试菌株的63.3%,Boty型菌株占供试菌株的28.3%,Flipper型菌株最少,仅占8.4%,未检测到vacuma型菌株。选取属于不同转座子类型的18株菌株测定其对草莓叶片的致病力,结果显示Boty型菌株所致病斑的平均直径显著大于Flipper型。草莓灰霉病菌转座子类型与致病力的关系有待进一步研究。转座子类型的检测为进一步研究灰葡萄孢的遗传多样性及遗传变异提供了基础。     

Hymenopteran Parasitoids on Fruit-infesting Tephritidae (Diptera) in Latin America and the Southern United States: Diversity, Distribution, Taxonomic Status and their use in Fruit Fly Biological Control

We first discuss the diversity of fruit fly (Diptera: Tephritidae) parasitoids (Hymenoptera) of the Neotropics. Even though the emphasis is on Anastrepha parasitoids, we also review all the information available on parasitoids attacking flies in the genera Ceratitis, Rhagoletis, Rhagoletotrypeta, Toxotrypana and Zonosemata. We center our analysis in parasitoid guilds, parasitoid assemblage size and fly host profiles. We also discuss distribution patterns and the taxonomic status of all known Anastrepha parasitoids. We follow by providing a historical overview of biological control of pestiferous tephritids in Latin American and Florida (U.S.A.) and by analyzing the success or failure of classical and augmentative biological control programs implemented to date in these regions. We also discuss the lack of success of introductions of exotic fruit fly parasitoids in various Latin American countries. We finish by discussing the most pressing needs related to fruit fly biological control (classical, augmentative, and conservation modalities) in areas of the Neotropics where fruit fly populations severely restrict the development of commercial fruit growing. We also address the need for much more intensive research on the bioecology of native fruit fly parasitoids.     

Brief Guide for Authors

正Journal of Arid Land(JAL)is an international journal(ISSN 1674-6767;CN 65-1278/K)for the natural sciences,sponsored by the Xinjiang Institute of Ecology and Geography,Chinese Academy of Sciences and Science Press.It is published by Science Press and Springer-Verlag Berlin Heidelberg bimonthly.JAL publishes original,innovative,and integrative research from arid and semiarid regions,ad     

The parasitoid complex ofLiriomyza cicerina on chickpea (Cicer arietinum)

Liriomyza cicerina (Diptera: Agromyzidae) is an important pest on chickpea in Turkey. The objective of this study was to determine the parasitoids and rates of parasitism ofL. cicerina on chickpea (Cicer arietinum L.) during the 2005 and 2006 seasons in ?anl?urfa province, Turkey. Leaves with mines were sampled weekly and kept in the laboratory to observe and count emerging leafminer and parasitoid adults. Eight parasitoid species were collected: the braconidsOpius monilicornis Fischer andOpius tersus Foerster and the eulophidsDiaulinopsis arenaria (Erdös) andNeochrysocharis formosa (Westwood), which occurred in both the winter and summer seasons;Diglyphus crassinervis Erdös,Neochrysocharis ambitiosa Hansson,Neochrysocharis sericea (Erdös) andPediobius metallicus (Nees), which occurred only in the summer growing areas.Diaulinopsis arenaria was the predominant parasitoid with 4–7.7% parasitism rate whileN. ambitiosa andO. monilicornis were the second and third most predominant species. The results of these trials show that sinceDia. arenaria occurred throughout every season, it could potentially be used for control of the leafminerL. cicerina.     

Plant–herbivore asynchrony necessitates augmentative releases of the exotic beetle,Zygogramma bicolorata,to enhance the biological control of P arthenium hysterophorus

Systematic information on the quantitative impact of Z ygogramma bicolorata on the biology of P arthenium hysterophorus is crucial as the seeds of this weed continue to germinate from the accumulated soil seed bank throughout the year in the form of different germinating flushes, while the activity of the beetle ceases during winter as it enters diapause. Therefore, plant–herbivore interactions need to be explored to develop predictions of the overall impact of the introduced beetle on the weed. The findings revealed that defoliation by Z . bicolorata had a significant impact on the plant height, density and flower production in flushes F ₃, F ₄ and F ₅, but not in F ₁ and F ₂ that exhibited longer periodicity, profuse branching, a longer flowering period and maximum flower production and contributed mostly to the existing seed soil bank. Therefore, total depletion of the existing soil seed bank was not possible. Consequently, the effect of augmentative field releases of laboratory‐reared beetles was explored on F ₁ and F ₂ in February for three consecutive years (2011–2013). Before initiating the trial, random soil samples were taken from the plots that were assigned to the paired treatments (i.e. with the beetle and without the beetle [insecticide‐treated]) and it was found that the seed bank in those samples did not differ. The single release of Z . bicolorata adults at five per plant at the six‐leaf stage significantly reduced the soil seed bank, compared to without the biocontrol agent, irrespective of the flushes at the end of the season.     

Effects of the saprophytic leaf mycoflora on growth and productivity of winter wheat

The effects of the saprophytic mycoflora and its interference with cereal aphids on growth and yield of winter and spring wheat was studied in field experiments in 1980, 1981 and 1982.Yields varied between 5000 and 8000 kg dry matter of kernels per ha. The effect of the saprophytic mycoflora on yield was determined in different treatments: A) no control measures against cereal aphids and saprophytic and necrotrophic fungi, B) no control of cereal aphids, control of saprophytic and necrotrophic fungi, C) control of cereal aphids and control of saprophytic and necrotrophic fungi, D) control of cereal aphids and stimulation of saprophytic mycoflora and E) control of cereal aphids, no control of saprophytic and necrotrophic fungi nor stimulation of saprophytic mycoflora.Considerable differences in top densities of saprophytic mycoflora (10 times as large in A and D as in B and C) were determined. The consequences of these differences for the growth and productivity of wheat were minor. A negative effect of saprophytic mycoflora on the yield could not be detected in 1981 and 1982, whereas a small positive significant effect was found in 1980. This stimulation may have been due to competition between necrotrophic fungal pathogens and saprophytic mycoflora. As a result of favourable weather conditions necrotrophic fungal pathogens were very numerous in 1980 and could form an important yield reducing factor. Yield levels may effect the importance of the necrotrophic and saprophytic mycoflora as yield reducing factors. Additionally, in the presence of aphid honeydew captafol was found to be relatively ineffective against saprophytic fungi.     

Relationship Between Production of the Phytotoxin Prehelminthosporol and Virulence in Isolates of the Plant Pathogenic Fungus Bipolaris sorokiniana

A gas chromatographic method was developed to quantify the phytotoxin prehelminthosporol, which is a sesquiterpene metabolite of the plant pathogen Bipolaris sorokiniana. The toxin was extracted from mycelium or culture filtrates, pre-cleaned using solid phase extraction, and analyzed by gas chromatography as a trimethylsilyl-derivative. The detection limit of the method was 5ngl^–1 (signal to noise ratio 4:1) which corresponds to ca. 15ng prehelminthosporol per mg dry weight of mycelium or 15ng prehelminthosporol per ml culture filtrate. The total amount of prehelminthosporol (mycelium plus culture filtrate) increased with cultivation time when examined in six isolates of B. sorokiniana after 6, 9, 12 and 15 days of incubation. The screening experiment of 17 isolates for prehelminthosporol production after 8 days of incubation revealed significant differences in the toxin production between the isolates. The isolates with low toxin production had lower virulence towards barley roots compared to those with higher production of the toxin. However, the virulence did not increase with prehelminthosporol level among the high producing isolates. Prehelminthosporol was also analyzed in a number of related Bipolaris and Drechslera species. In addition to B. sorokiniana, three out of six Bipolaris species (B. setariae, B. zeicola, B. victoriae) produced prehelminthosporol, which indicates that ability to produce prehelminthosporol is conserved among closely-related Bipolaris species.     

Effect of botanical insecticides and bacterial toxins on the gut enzyme of the rice leaffolderCnaphalocrocis medinalis

The effect of botanical insecticides and bacterial toxins on gut enzyme activity of larvae of the rice leaffolderCnaphalocrocis medinalis (Guenée) (Insecta: Lepidoptera: Pyralidae) was investigated. Gut enzyme activities were affected by botanical insecticides and bacterial toxin individually and in combination. When fed a diet of rice leaves treated with botanical insecticides and bacterial toxins, in bioassays the activities of gut tissue enzymes — acid phosphatases (ACP), alkaline phosphatases (ALP) and adenosine triphosphatases (ATPase) — of rice leaffolder larvae were affected. When combined, the effect was more severe at a low concentration. Larvae that were chronically exposed to botanical insecticides and bacterial toxins showed a reduction in weight (59–89%) and exhibited a significant reduction in ACP, ALP and ATPase activities. The combination ofBacillus thuringiensis kurstaki and botanical insecticides caused a decrease of twofold in enzyme activity even at reduced concentration. A synergistic effect was found when botanical insecticides and bacterial toxins were combined at low doses. These effects were most pronounced in early instars. Clear dose-response relationships were established with respect to enzyme activity. In conclusion: (i) biopesticides are relatively safe and biodegradable; (ii) a synergistic effect of botanical insecticides and bacterial toxins was found; (iii) less expensive, readily available and naturally occurring biopesticides could be an alternative for organic and inorganic pesticides in controlling RLF. http://www.phytoparasitica.org posting Sept. 28, 2004.