Genetic and pathogenic diversity within Ascochyta rabiei(Pass.) Lab. populations in Pakistan causing blight of chickpea (Cicer arietinum L.)

Pathogenic and genetic diversity in Ascochyta rabiei populations in Pakistan were evaluated. Biological pathotyping of 130 A. rabiei isolates (obtained from hierarchically collected samples) was conducted on a set of three chickpea differentials, i.e. ILC 1929 (susceptible), ILC 482 (tolerant) and ILC 3279 (resistant), under controlled conditions. Disease severity data were recorded 12 days after inoculation. Statistical analysis grouped the isolates into three pathotype classes. Four isolates belonged to pathotype I (least aggressive), 79 isolates to pathotype II (medium aggressive) and 47 isolates to pathotype-III (highly aggressive).Genetic analysis was performed using RAPDs and oligonucleotide fingerprinting, where Hinf I-digested DNA was hybridized to the³²P-endlabeled oligonucleotide probes (CAA)₅, (GAA)₅, (GA)₈, (CA)₈and (GATA)₄. Dendrograms produced by cluster analysis discriminated 46 genotypes in the A. rabiei population of Pakistan. Genetic distances and relatedness between isolates were calculated. At a genetic distance of 0.3, genotypes were divided into six distinct genotype groups A, B, C, D, E and F containing 16, 11, 2, 5, 5 and 7 isolates, respectively. Most of the genotypes were area specific or predominated in certain areas but did not belong to a distinct pathotype, while most of the aggressive isolates (pathotype III) occurred in Northern Punjab and in the North Western Frontier Province.     

壳二孢叶枯病病原菌Ascochyta rabiei胁迫下鹰嘴豆防御相关基因的表达分析

为挖掘获得新的抗性基因,培育鹰嘴豆Cicer arietinum抗壳二孢叶枯病(病原菌为Ascochyta rabiei)新品种,以项目组前期获得的102个差异表达的新基因为基础,随机选取29个基因进行同源性分析,以鹰嘴豆Actin(EU529707)和Ef-1α(AJ004960)作为参考基因,利用实时荧光定量PCR技术检测这29个基因在宿主植物鹰嘴豆抗性品种系选03中的表达规律。结果显示,基于同源性分析结果可将29个基因大致分成4类,涉及信号传导机制、细胞运输、转录和细胞拯救、防御、毒性;功能分析结果显示,功能未知的基因数目最多,达到了11个,其中多数为鹰嘴豆未定性基因。这29个基因在A. rabiei胁迫下都发生了不同程度的差异表达,表达差异时间点集中在胁迫后72 h,并在96 h恢复至正常表达水平。其中解毒相关基因474在72 h时相对表达水平最高,是对照处理0 h的19.773倍,抗氧化修复相关基因1137的相对表达水平最低,约为对照处理0 h的1/3。筛选获得的差异表达基因中,表达上调的基因有10个,表达下调的基因有16个,其余3个基因的表达差异不明显。上调表达基因可能与鹰嘴豆应对A. rabiei侵染的应答机制有关,其中与免疫应激相关的蛋白基因如谷胱甘肽S-转移酶、咖啡酰辅酶A、泛素蛋白基因等可能直接参与了鹰嘴豆应对A. rabiei的免疫识别和防御。     

Development of ascochyta blight (Ascochyta rabiei) in chickpea as affected by host resistance and plant age

Ascochyta blight caused by Ascochyta rabiei, is the most destructive disease in many chickpea growing countries. Disease development varies with the growth stage and host resistance. Hence, disease development was studied in cvs ICCX 810800 (resistant), ICCV 90201 (moderately resistant), C 235 (moderately susceptible), ICCV 96029 and Pb 7 (susceptible) under controlled environment (ICRISAT, Patencheru) and field conditions (Dhaulakuan, Himachal Pradesh) at seedling, post-seedling, vegetative, flowering and podding stages. Under controlled environment, the incubation period and terminal disease reaction (TDR) did not vary significantly at different growth stages against virulent isolate AB 4. Cultivars ICCX 810800, ICCV 90201 and C 235 showed a significantly longer incubation period than the susceptible cv. Pb 7. Cultivar ICCX 810800 showed slow disease progress and the least TDR. Field experiments were conducted during the 2003–2004 and 2004–2005 growing seasons. During 2003–2004, TDR was higher in plants inoculated at podding and the flowering stage and the lowest disease reaction was recorded in ICCX 810800. A severe epidemic during 2004–2005 was attributed to the favourable temperature, humidity and well distributed high rainfall. TDR did not differ significantly at any of the growth stages in susceptible cvs ICCV 96029 and Pb 7. With respect to seeding date and cultivar, the highest yield was recorded in the early-sown crop (1,276.7 kg ha⁻¹) and in ICCV 90201 (1,799.3 kg ha⁻¹), respectively. The yields were greatly reduced in all the cultivars during 2004–2005 and the highest yield was recorded in ICCX 810800 (524.7 kg ha⁻¹). Integrated disease management using resistant cultivars, optimum sowing period and foliar application of fungicides will improve chickpea production. The experiment under controlled environment and field conditions (during the epidemic year) showed a similar disease development.     

Mating type, pathotype and RAPDs analysis inDidymella rabiei, the agent of ascochyta blight of chickpea

Eleven pathotype groups (A-K), including five not previously reported, ofDidymella rabiei (anamorphAscochyta rabiei), representing isolates of the pathogen from Ascochyta blight-affected chickpeas mainly from India, Pakistan, Spain and the USA, were characterized using 44 single-spore isolates tested against seven differential chickpea lines. Of 48 isolates tested for mating type, 58% belonged to MAT 1-1 and 42% to MAT 1-2. Thirty-nineD. rabiei isolates, as well as two isolates ofAscochyta pisi and six isolates of unrelated fungi, were analyzed using Randomly Amplified Polymorphic DNAs (RAPDs) employing five primers (P2 at 40°C, and OPA3, OPC1, OPC11 and OPC20 at 35°C). Computer cluster analysis (UPGMA / NTSYS-PC) detected a relatively low level of polymorphism among all theD. rabiei isolates, although atca 7% dissimilarity,ca 10 RAPD groups [I-X] were demarcated, as well as subclustering within the larger groups. By the same criteria, the maximum dissimilarity for the whole population ofD. rabiei isolates wasca 13%. No correlation was found between different RAPD groups, pathotype, or mating type ofD. rabiei, although some evidence of clustering based on geographic origin was detected. The use of RAPDs enabled us to identify specific DNA fragments that may have a potential use as genetic markers in sexual crosses, but none which could be used as virulence markers.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Validation of a QTL for resistance to ascochyta blight linked to resistance to fusarium wilt race 5 in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Ascochyta blight caused by Ascochyta rabiei and fusarium wilt caused by Fusarium oxysporum. f. sp. ciceris are the two most serious diseases of chickpea (Cicer arietinum). Quantitative trait loci (QTL) or genes for ascochyta blight resistance and a cluster of resistance genes for several fusarium wilt races (foc1, foc3, foc4 and foc5) located on LG2 of the chickpea map have been reported independently. In order to validate these results and study the linkage relationship between the loci that confer resistance to blight and wilt, an intraspecific chickpea recombinant inbred lines (RIL) population that segregates for resistance to both diseases was studied. A new LG2 was established using sequence tagged microsatellite sites (STMS) markers selected from other chickpea maps. Resistance to race 5 of F. oxysporum (foc5) was inherited as a single gene and mapped to LG2, flanked by the STMS markers TA110 (6.5 cM apart) and TA59 (8.9 cM apart). A QTL for resistance to ascochyta blight (QTL_AR3) was also detected on LG2 using evaluation data obtained separately in two cropping seasons. This genomic region, where QTL_AR3 is located, was highly saturated with STMS markers. STMS TA194 appeared tightly linked to QTL_AR3 and was flanked by the STMS markers TR58 and TS82 (6.5 cM apart). The genetic distance between foc5 and QTL_AR3 peak was around 24 cM including six markers within this interval. The markers linked to both loci could facilitate the pyramiding of resistance genes for both diseases through MAS.     

Defense strategies of pea embryo axes with different levels of sucrose to Fusarium oxysporum and Ascochyta pisi

The aim of this study was to compare the defense responses of embryo axes of Pisum sativum L. cv. Kwestor with different sucrose levels to pathogenic fungi, i.e. systemic acting Fusarium oxysporum f. sp. pisi and locally acting Ascochyta pisi. Embryo axes were cultured on Heller medium for 96 h. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). After inoculation of the pea embryo axes with pathogenic fungi a generally higher concentration of free radicals was detected by electron paramagnetic resonance (EPR), in comparison to non-inoculated embryo axes. The inoculation with F. oxysporum caused stronger generation of free radicals in −Si than in +Si embryo axes. A different response was observed after inoculation with A. pisi; starting from 48 h, the concentration of free radicals in +Si axes was found to be 1.5 times higher than in −Si embryo axes. The values of spectroscopic splitting coefficients for these radicals suggest that they are semiquinone radicals. The EPR method also revealed Mn²⁺ ion accumulation after 24 h of culture. Over time, high levels of these ions were recorded in +Si embryo axes inoculated with F. oxysporum, while in +Si embryo axes inoculated with A. pisi they decreased. Up to 48 h after inoculation with the pathogenic fungi, Mn²⁺ ion levels were higher in +Si embryo axes than in +Sn axes. The activity of superoxide dismutase (SOD, EC 1.15.1.1) increased in +Si embryo axes up to 72 h after inoculation with pathogenic fungi; however, it was generally lower than in +Sn axes. Catalase activity (CAT, EC 1.11.1.6) increased up to 72 h after inoculation with F. oxysporum and the values were higher than in the non-inoculated tissue. Especially high activity of this enzyme was noted in −Si embryo axes after inoculation with either F. oxysporum or A. pisi. Peroxidase activity (POX, EC 1.11.1.7) towards pyrogallol in embryo axes increased during culture; however, it was lower or similar to that in non-inoculated embryo axes. SOD, CAT and POX zymograms showed that the synthesis of new isoforms was induced after inoculation with pathogenic fungi. Peroxidase isozymes detected by the reaction with diaminobenzidine in native PAGE were intensely stained in +Si embryo axes after inoculation with pathogenic fungi. Respiratory activity of the inoculated tissues was considerably higher than in non-inoculated tissues. The respiration rate was generally much higher in +Si than in −Si embryo axes. Growth of −Si embryo axes was more significantly retarded as a consequence of inoculation than that of +Si embryo axes.These results indicate that, depending on the manner of influence of a pathogenic fungus, both similar and differing defensive strategies may be initiated and a raised sugar levels in pea tissues limit the development of F. oxysporum and A. pisi.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

Destruxin B produced by Alternaria brassicae does not induce accessibility of host plants to fungal invasion

It has been reported that Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, produces a host-specific or host-selective toxin (HSTs) identified as destruxin B. In this study, the role of destruxin B in infection of the pathogen was investigated. Destruxin B purified from culture filtrates (CFs) of A. brassicae induced chlorosis on host leaves at 50–100 μg ml⁻¹, and chlorosis or necrosis on non-host leaves at 250–500 μg ml⁻¹. Destruxin B was detected in spore germination fluids (SGFs) on host and non-host leaves, but not in a sufficient amount to exert toxicity to host plants. When spores of non-pathogenic A. alternata were combined with destruxin B at 100 μg ml⁻¹ and inoculated on the leaves, destruxin B did not affect the infection behavior of the spores. Interestingly, SGF on host leaves allowed non-pathogenic spores to colonize host leaves. Moreover, a high molecular weight fraction (>5 kDa) without destruxin B obtained by ultrafiltration of SGF had host-specific toxin activity and infection-inducing activity. From these results, we conclude that destruxin B is not a HST and does not induce the accessibility of the host plant which is essential for colonization of the pathogen. In addition, the results with SGF imply that a high molecular weight HST(s) is involved in the host–pathogen interaction.     

Suppression of Rhizoctonia root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated with their effect on the pathogen growth and on the root morphogenesis

Rhizoctonia solani root-rot is a major soilborne disease causing growth and yield depression. The ability of Glomus mosseae BEG12 and Pseudomonas fluorescens A6RI to suppress this soilborne disease in tomato was assessed by comparing the shoot and root growth of plants infested with R. solani 1556 when protected or not by these beneficial strains. The epiphytic and parasitic growth of the pathogenic R. solani 1556 was compared in the presence and absence of the biocontrol agents by microscopical observations allowing the quantification of roots with hyphae appressed to epidermal cells (epiphytic growth) and of roots with intraradical infection (parasitic growth). The root architecture of the tomato plants under the different experimental conditions was further characterized by measuring total root length, mean root diameter, number of root tips and by calculating degree of root branching. G. mosseae BEG12 and P. fluorescens A6RI fully overcame the growth depression caused by R. solani 1556. This disease suppression was associated with a significant decrease of the epiphytic and parasitic growth of the pathogen together with an increase of root length and of the number of root tips of inoculated tomato plants. The combined effects of G. mosseae BEG12 and P. fluorescens A6RI on pathogen growth and on root morphogenesis are suggested to be involved in the efficient disease suppression.     

Glomus intraradices, Pseudomonas alcaligenes, and Bacillus pumilus: effective agents for the control of root-rot disease complex of chickpea (Cicer arietinum L.)

The effects of Glomus intraradices, Pseudomonas alcaligenes and Bacillus pumilus on the root-rot disease complex caused by the root-knot nematode Meloidogyne incognita and the root-rot fungus Macrophomina phaseolina in chickpea was assessed by quantifying differences in the shoot dry mass, pod number, nodulation, and shoot content of chlorophyll, nitrogen, phosphorus and potassium. Inoculation of plants with G. intraradices, P. alcaligenes and B. pumilus alone and in combination significantly increased shoot dry mass, pod number, and content of chlorophyll, nitrogen, phosphorus and potassium in plants inoculated with pathogens over that in the uninoculated control plants. P. alcaligenes caused a greater increase in shoot dry mass, pod number, chlorophyll, nitrogen, phosphorus and potassium in plants with pathogens than did G. intraradices or B. pumilus. Combined application of G. intraradices, P. alcaligenes and B. pumilus to plants inoculated with pathogens caused a greater increase in shoot dry mass, pod number, nitrogen, phosphorus, and potassium than did an application of P. alcaligenes plus B. pumilus or of G. intraradices plus B. pumilus. Root colonization by G. intrardices was high when used alone, while inoculation with the pathogens reduced root colonization by G. intraradices. In the presence of P. alcaligenes and B. pumilus, root colonization by G. intraradices increased. In plants inoculated with just one antagonist, P. alcaligenes reduced galling and nematode multiplication the most, followed by G. intraradices, then B. pumilus. The greatest reduction in galling, nematode multiplication and root-rot was observed when both bacterial species and G. intraradices were applied together.     

口红吊兰菌核病病原鉴定及其生物学特性分析

为鉴定1株分离自口红吊兰叶部病斑上的疑似核盘菌菌株,利用柯赫氏法则验证其致病性并通过形态观察和ITS序列分析对该病原菌进行分类鉴定,结合温度、酸碱度等生理指标研究其生物学特性,并利用菌丝生长速率法测定了扑海因、多菌灵、苯醚甲环唑、腐霉利4种药剂对该病原菌的抑制作用。结果显示:在PDA平板上该病原菌菌丝为白色,均匀生长;约7 d后开始产生菌核,直径3~5 mm;菌核萌发后可产生1~3个子囊盘,内含8个大小为8.0~12.0μm×4.0~5.5μm的子囊孢子。该菌株ITS序列系统进化分析结果显示,其与核盘菌的同源性高达99%。综上所述,初步确定该菌株为核盘菌Sclerotinia sclerotiorum。该病原菌在PDA培养基上的最适生长温度为20~25℃、最适生长p H为5~7。室内毒力测定发现供试4种药剂中扑海因对该病原菌菌丝生长有较好的抑制效果,且其EC_(50)最小,仅为0.62 mg/L,证明扑海因对核盘菌毒力最强。     

植物病原棒形杆菌属分类最新进展

植物病原棒形杆菌属Clavibacter的分类随着研究的深入和鉴定技术水平的不断进步一直在发生着变化,之前普遍认可的观点为该属仅含有1个种,即密执安棒形杆菌C. michiganensis,种下又分为5个亚种,分别为密执安亚种C. michiganensis subsp. michiganensis、诡橘亚种C. michiganensis subsp. insidiosus、尼布拉斯加亚种C. michiganensis subsp. nebraskensis、环腐亚种C. michiganensis subsp. sepedonicus和花叶亚种C. michiganensis subsp. tessellarius。最近几年又有4个新亚种陆续被报道,分别为菜豆亚种C. michiganensis subsp. phaseoli、辣椒亚种C. michiganensis subsp. capsici、加利福尼亚亚种C. michiganensis subsp. californiensis和智利亚种C. michiganensis subsp. chilensis。随着全基因组分析和多基因分析技术在细菌分类上的应用,之前被广泛认可的4个亚种(诡橘亚种、尼布拉斯加亚种、环腐亚种和花叶亚种)及新亚种辣椒亚种也曾被建议定义为种,即C. insidiosus、C. nebraskensis、C. sepedonicus、C. tessellarius和C. capsici。为便于研究人员了解该属最新的分类研究现状,本文对植物病原棒形杆菌属的分类历史及最新分类现状进行了系统梳理,期望为该属病原菌的风险评估、检测鉴定等研究提供分类学参考。     

芥菜乙醇提取物对西瓜枯萎病菌的抑菌机制

为探究芥菜乙醇提取物对植物病原菌的抑制机理,以西瓜枯萎病菌Fusarium oxysporum f.sp. niveum为指示菌,以最低抑菌浓度(minimum inhibitory concentration,MIC)0.5 g/mL为基准,设置0、0.25、0.5和1.0 g/mL共4种处理浓度,通过测定菌丝形态结构、菌液电导率、菌体丙二醛含量、细胞壁相关水解酶活性、呼吸代谢途径酶活性、可溶性蛋白含量及还原糖含量等指标的变化分析其抑菌机理。结果显示,1.0 g/mL芥菜乙醇提取物对西瓜枯萎病菌的处理效果最好,其中,处理后病菌菌液的电导率明显增大,最大可达1.45 ms/cm,是对照组的1.57倍;丙二醛含量达3.16 mmol/g,为对照组的2.24倍;几丁质酶和β-1,3-葡聚糖酶的活性最高,分别为10.57 U/g和14.16 U/g,较对照组分别显著升高了48.04%和17.80%,同时细胞壁降解,使细胞完整性受损;琥珀酸脱氢酶和苹果酸脱氢酶的活性最低,分别为7.33 U/mg和0.28 U/mg,较对照组分别显著下降了73.02%和47.17%;可溶性蛋白和还原糖的含量显著降低,分别为0.44 mg/g和5.34 mg/g,较对照组分别下降了60.71%和56.30%,呼吸能量代谢受阻,抑制菌体生长。表明芥菜乙醇提取物可有效防治西瓜枯萎病。     

辣木枝枯病病原菌鉴定及其生物学特性

为明确海南省辣木枝枯病病原菌及其生物学特性,采用组织分离法获得菌株MO157,通过柯赫氏法则、形态学特征及分子生物学技术对该菌株进行鉴定,并测定了其菌丝生长的最佳条件。结果表明,菌株MO157回接10 d后,辣木茎干表面周围呈黄褐色,随后发病部位表现出水渍状病斑,与自然发病辣木的病状相符;菌落近圆形,菌丝初期呈白色绒毛状,后期呈浅黄色,分生孢子顶胞钩状,3~5个隔膜,厚垣孢子呈球形,在菌丝间串生;菌株MO157的ITS序列与Gen Bank中木贼镰刀菌Fusarium equiseti的相似性为100%,结合形态特征与分子鉴定最终将其确定为木贼镰刀菌(Gen Bank登陆号:KX197955)。该菌菌丝生长适宜温度25~30℃,最适温度28℃;pH 6~8时菌丝生长速率增快,pH 7时最适菌丝生长;PDA培养基和PSA培养基最适合该菌生长,以蔗糖为碳源时利用率最高;以牛肉浸膏为氮源时利用率最高;该菌致死温度为65℃,10 min;光照对菌丝生长影响较小,不同处理间菌落直径无显著差异。     

Relative tolerances of wild and cultivated barley to infection by Blumeria graminis f.sp. hordei (Syn. Erysiphe graminis f.sp. hordei). II—the effects of infection on photosynthesis and respiration

The relative levels of tolerance of two wild barley lines (Hordeum spontaneum), B19909 and I-17-40, and one cultivated barley (Hordeum vulgare), cv. Prisma, to Blumeria graminis f.sp. hordei were determined by comparing the effects of different levels of infection on the photosynthesis and respiration rates of the third leaf. Infection caused the early onset of senescence in all three lines, and in particular in cv. Prisma, and was accompanied by decreases in gross and net photosynthesis rates, increases in respiration rates, and loss of chlorophyll. The onset of senescence occurred at approximately the same time in infected leaves of the two wild lines, but once triggered, photosynthesis rates and chlorophyll levels declined more rapidly in I-17-40 than in B19909. A burst of respiratory activity accompanied the onset of senescence, and this was greatest in cv. Prisma. Conidial production was higher in B19909, indicating a higher level of tolerance in this line.     

苦皮藤素V在粘虫和小地老虎幼虫体内的穿透与代谢

采用高效液相色谱技术研究了苦皮藤素Ⅴ在粘虫Mythimna separata和小地老虎Agrotis ypsilon幼虫中的穿透及代谢。结果表明:苦皮藤素V均不能从粘虫和小地老虎幼虫的体壁穿透到血腔或从血腔穿透到中肠,但很容易从中肠穿透到血腔,且穿透速率无差异;苦皮藤素V在小地老虎幼虫体内的代谢解毒速率远大于其在粘虫幼虫体内的代谢速率,其半衰期分别为5.5和13.1 h。本研究结果表明,苦皮藤素V对粘虫和小地老虎幼虫的选择毒杀作用与药物的穿透能力无关,其在试虫体内的解毒代谢差异才是其对昆虫具有选择作用的机理之一。     

甘蔗赤条病菌巢式PCR检测

甘蔗赤条病是由燕麦食酸菌燕麦亚种(Acidovorax avenae subsp.avenae,Aaa)引起的一种世界性甘蔗细菌病害。为建立Aaa快速、灵敏的检测技术,根据该病菌16S~23S核糖体基因及其转录间隔区ITS分别设计2对特异性引物,建立Aaa巢式PCR检测方法。结果表明,建立的巢式PCR方法对Aaa标准菌株、水稻食酸菌A.oryzae具有特异性,可扩增出454 bp目的条带,对近缘种德氏食酸菌A.delafieldii及其它科属的红色雷夫松氏菌Leifsonia rubra和甘蔗宿根矮化病菌L.xyli subsp.xyli未扩增出任何条带。以感染Aaa的甘蔗叶片总DNA、含ITS靶标片段的质粒DNA标准品及Aaa标准菌液为模板,巢式PCR灵敏度最低检测限分别为10 fg/μL、10拷贝/μL和36 CFU/mL,是常规PCR灵敏度的1 000倍。应用巢式PCR和常规PCR对14份有赤条病症状的田间甘蔗叶片样品进行平行检测,阳性检出率分别为100.0%和28.6%,表明巢式PCR比常规PCR检测方法具有更高的灵敏度。本研究建立的巢式PCR方法适合于田间甘蔗赤条病害的分子检测与鉴定。