Decrease of rice plant resistance and induction of hormesis and carboxylesterase titre in brown planthopper, Nilaparvata lugens (Stål) by xenobiotics

The brown planthopper (BPH), Nilaparvata lugens (Stål) is a serious threat to the rice production throughout Asia. The indiscriminate application of various xenobiotics in rice ecosystem is perceived as one of the factors for the frequent outbreak of BPH. The present study has critically analysed the secondary effects of some xenobiotics used in rice field on certain plant and insect parameters that subsequently favour BPH outbreak. Application of 2,4-D, carbendazim, deltamethrin and urea reduced the innate BPH resistance of PTB 33 rice variety due to favourable alterations in rice free amino acid and sucrose content. Similarly, these chemicals also induced hormesis and enhanced feeding in BPH. Alternatively, soil amendment with neem seed powder and Calotropis gigantea leaves improved plant innate resistance and showed no sign of hormesis or enhanced feeding in BPH. In addition, deltamethrin has the ability to stimulate BPH carboxylesterase titre. Native PAGE analysis of esterases from whole body homogenate of BPH revealed at least five esterase isozyme bands, prominent being E1 and E2. However, no difference in BPH esterase banding pattern was observed between different xenobiotic treatments. All these esterase bands are classified under carboxylesterase based on their inhibition by class specific esterase inhibitors.     

Molecular analysis of resistance in a deltamethrin-resistant strain of Musca domestica from China

We investigated the molecular basis of resistance in a strain of house fly (BJD) from Beijing, China. This strain showed 567-fold resistance to commonly used deltamethrin. Flies were 64-fold resistant to deltamethrin synergized by piperonyl butoxide (PBO). The 5′-flanking sequence of the cytochrome P450 gene CYP6D1 in BJD strain had a 15-bp insert as in the LPR strain. Two mutations (kdr, super-kdr) in the voltage sensitive sodium channel (VSSC) were also detected in the BJD strain. Our results showed that a combination of resistance alleles for CYP6D1 and VSSC existed in deltamethrin resistant house flies in China.     

Chromosome and plasmid-encoded N-acyl homoserine lactones produced by Agrobacterium vitis wildtype and mutants that differ in their interactions with grape and tobacco

Agrobacterium vitis causes crown gall disease on grapevines. It also induces a specific necrosis on grape roots and a hypersensitive response (HR) on tobacco that are regulated by a complex quorum-sensing regulatory system. Strain F2/5 produces at least six N-acyl-homoserine lactones (AHLs) that function as signal molecules in quorum-sensing. The AHLs differ in acyl side chain length (8–16 carbons) as determined by gas chromatography/mass spectrometry and electrospray ionization tandem mass spectrometry. Mutant derivatives of F2/5 differ in ability to cause necrosis and the HR and show variable AHL profiles as determined by a thin-layer chromatography/biosensor assay. All wildtype A. vitis strains revealed the presence of long-chain AHLs regardless of tumorigenicity or ability to cause the HR. Whereas genes encoding long-chain AHLs are predicted to reside on the F2/5 chromosome, the determinants for short-chain AHLs were shown to be located on conjugal plasmids.     

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Acacia farnesiana lectin-like protein (AFAL) showed bacterioestatic effects against Xanthomonas axonopodis pv. passiflorae (Gram-negative) and Clavibacter michiganensis michiganensis (Gram-positive), with the latter being more sensitive. This effect is probably due to the ability of AFAL to interact with the bacterial cell wall where we observed that AFAL induced macroscopic change. The maximum bacterial growth inhibition was approximately 78% when incubated with Gram-negative strains, and as high as 92% percent for the Gram-positive one. The antibacterial effect of flavonoids (rutin, quercetin and morin) was also observed using low concentrations against both bacterial strains. Prior incubation of both with AFAL at high concentrations increases the inhibitory effect of flavonoids on bacterial growth. The potential use of AFAL as a control agent against the root-knot nematode Meloidogyne incognita was investigated as well, showing anti-nematode properties involving both egg hatching and motility. In the juvenile second-stage, AFAL showed reduction in larval mobility when measured against a control group. The results suggest that AFAL is effective against M. incognita and could be used as a component of integrated pest management programs. These data also suggest that lectins probably play a role in plant defense not only against invertebrate phytopathogens, herbivores and fungi but also against bacteria.     

High resistance to rice yellow mottle virus in two cultivated rice cultivars is correlated with failure of cell to cell movement

Rice yellow mottle virus (RYMV) accumulation in protoplasts and whole plants was investigated in two highly resistant cultivars, Tog5681 (Oryza glaberrima) and Gigante (Oryza sativa). Three susceptible cultivars, i.e. one O. glaberrima Tog5673 and two O. sativa (IR64, Ac. 2428), and a partially resistant cultivar (Azucena) were used as control. After inoculation, accumulation of coat protein (CP) and viral RNA were monitored on protoplasts, inoculated leaves, sheaths of inoculated leaves and newly infected leaves by serological and Northern blot analysis. Viral RNA accumulated to a similar extent in protoplasts from all cultivars studied. In contrast, three distinct in planta behaviors were noted. In susceptible plants (IR64, Tog5673 and Ac. 2428), there was high CP and RNA accumulation at 5 d.p.i. in whole plants, suggesting that cell to cell and vascular movements occurred before 5 d.p.i. in inoculated leaves. The second behavior concerned Azucena, which showed a delay (around 7 d.p.i.) of viral accumulation in inoculated leaves. The third behavior involved the highly resistant cultivars Tog5681 and Gigante. CP and viral RNA were not detected in these cultivars. The comparison of viral accumulation in protoplasts and plants suggested that resistance of the highly resistant cultivars Tog5681 (O. glaberrima) and Gigante (O. sativa) was not due to the inhibition of virus replication but rather to the failure of cell to cell movement.     

Enhancing resistance of transgenic carrot to fungal pathogens by the expression of Pseudomonas fluorescence microbial factor 3 (MF3) gene

Microbial factor 3 (MF3) gene from a plant-growth promoting rhizobacteria Pseudomonas fluorescence was introduced into carrot genome using Agrobacterium-mediated transformation. The obtained transgenic plants expressing MF3 protein were grown in a glasshouse and evaluated for their resistance to phytopathogens. The assays were performed by the assessment of disease symptoms developing on the detached leaflets and petioles inoculated by three fungal pathogens. The results showed that transgenic plants had significantly enhanced resistance to Alternaria dauci, Alternaria radicina and Botrytis cinerea, on average, by 20–40% in comparison to the non-transformed control plants. This is the first report of enhancing plant resistance by expressing the bacterial protein homologous to the family of FK506-binding proteins.     

Efficacy of certain agrochemicals on Aspergillus spp. and subsequent aflatoxin production in rice

Efficacy of certain fungicides and non-conventional chemicals against Aspergillus spp. contamination and subsequent aflatoxin production in rice was investigated. Among the 10 fungicides tested, carbendazim, contaf plus, folicur, propiconazole and saaf completely inhibited the growth of all Aspergillus spp. and aflatoxin B₁ (AFB1) production at 1 g or ml/kg concentration. Of the five non-conventional chemicals tested, benzoic acid effectively inhibited the mycelial growth of Aspergillus flavus (72%) at 4 g/kg, completely inhibited the Aspergillus parasiticus and Aspergillus niger even at 1 g/kg and Aspergillus ochraceus at 4 g/kg concentration. Vanillin completely reduced the AFB1 production at 4 g/kg of seed followed by sodium chloride with out inhibiting the mycelial growth. This study reveals that fungicides and non-conventional chemicals had effectively inhibited the mycelial growth of Aspergillus spp. and AFB1 production in rice.     

江苏省水稻赤霉病病原菌分离鉴定与致病力分析

为明确江苏省水稻赤霉病的病原菌组成及致病特征,于2018—2019年从江苏省13个市41个县区采集341份水稻样品,采用组织分离法对病原菌进行分离纯化,基于翻译延伸因子（translationelongation factor-1α,TEF-1α）序列分析对分离菌株进行鉴定,通过TRI11基因序列鉴定及产毒力测定对其产毒化学型进行分析,并按照柯赫氏法则对其致病力进行验证。结果显示,从水稻样品中共分离获得病原菌392株,其中亚洲镰刀菌Fusarium asiaticum为385株,禾谷镰刀菌F. graminearum为7株。亚洲镰刀菌分为3-乙酰化脱氧雪腐镰刀菌烯醇（3-acetyldeoxynivalenol,3ADON）化学型和雪腐镰刀菌烯醇（nivalenol,NIV）化学型菌株,分别占亚洲镰刀菌总菌株数的 66.8% 和 33.2%;禾谷镰刀菌均为（15-acetyldeoxynivalenol,15ADON）化学型。产3ADON的亚洲镰刀菌在全省范围内为优势群体,而产15ADON的禾谷镰刀菌仅在淮北地区被发现;产NIV的亚洲镰刀菌对水稻的致病性要显著高于产3ADON的亚洲镰刀菌。不同产毒化学型亚洲镰刀菌对水稻均有一定致病性,但NIV化学型亚洲镰刀菌对水稻的致病力最强。     

山东省小麦赤霉病菌种群组成及其致病力分化

由禾谷镰孢菌群Fusarium graminearum clade引起的赤霉病是小麦的重要病害。为明确山东省小麦赤霉病菌的种群组成及其致病力,于2011年和2012年从山东省15地市分离了95株小麦赤霉病菌,在形态和分子生物学鉴定种的基础上,采用鉴定B型毒素化学型的特异性引物进行毒素化学型分析。在95个菌株中,93株分离物为禾谷镰孢菌F.graminearum,2株为燕麦镰孢菌F.avenaceum。94株分离物为脱氧雪腐镰孢菌烯醇(deoxynivalenol,DON)化学型,1株为雪腐镰孢菌烯醇(nivalenol,NIV)化学型。在94株DON毒素化学型菌株中,90株为15-乙酰脱氧雪腐镰孢菌烯醇(15-acetyldeoxynivalenol,15-AcDON)化学型,4株为3-乙酰脱氧雪腐镰孢菌烯醇(3-acetyldeoxynivalenol,3-AcDON)化学型。在小麦扬花期,采用单花滴注接种法对29个菌株进行了致病力测定,供试菌株的致病力分化明显。表明在山东省冬小麦产区,产15-AcDON毒素的F.gra-minearum是小麦赤霉病菌的优势种群。     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Spontaneous ultraweak photon emission from rice (Oryza sativa L.) and paddy weeds treated with a sulfonylurea herbicide

All living organisms spontaneously generate ultraweak photon emissions, which originate from biochemical reactions in cells. Current research uses the ultraweak photon emissions from organisms as a novel tool to investigate the physiological states of plants. In this study, we found ultraweak photon emissions from leaf segments of rice and several paddy weed species treated with a sulfonylurea herbicide. There is a definite difference in photon emissions among plant species, and rice (Oryza sativa), barnyardgrass (Echinochloa crus-galli) and Cyperus serotinus showed extremely strong enhancement of photon emissions. Photon emissions from these three species treated with sulfonylurea herbicide were suppressed when the leaf segments were treated with the cytochrome P450 monooxygenase (P450) inhibitors, piperonyl butoxide and malathion. These results suggest that P450 inhibitors affect the ultraweak photon emissions from plants.     

Resistance development in rice blast disease caused by Magnaporthe grisea to tricyclazole

Sensitivity to tricyclazole of 129 single-conidial isolates of rice blast fungus, Magnaporthe grisea, was determined. EC₅₀ values ranged from 0.06 to 1.12 mg/L with an average value of 0.46 ± 0.09 mg/L according to the detached leaf segment tests. No significant difference of sensitivity was observed between isolates from Guangdong and Jiangsu where decreased efficacy was reported, and from two other provinces where tricyclazole provided excellent disease control. In seedling tests, tricyclazole could control the most tolerant isolate GY-6 successfully with a efficacy of 81.5% at the concentration of 40 mg/L. Sensitivities of GY-6 and DY-2, the most sensitive isolate, to tricyclazole were both unstable in sub-cultured single-conidial offspring isolates, with respective mean EC₅₀ values of 5.40 ± 0.97 and 4.50 ± 0.88 mg/L calculated from seedling tests. There was no amino acid difference between them in the coding sequences of 1,3,6,8-tetrahydroxynaphthalene reductase and 1,3,8-trihydroxynaphthalene reductase. These results suggested that the decreased control reported in Guangdong and Jiangsu could not be attributed to the occurrence of resistance. When continuously “inoculated-reisolated-reinoculated” under the selection of tricyclazole in vivo, sensitivity of DY-2 decreased 10-fold after 20 generations, although the sensitivity of GY-6 did not shift significantly.     

大麦赤霉病鉴定方法与抗性评价

为探讨以病小穗率与病穗率作为抗性评价指标的精确度,利用单花滴注、孢子液喷雾分别结合土表病麦粒接种的方法,评价了2个大麦重组自交系群体131个株系对赤霉病的抗侵染与抗扩展性。单花滴注接种后调查了第7、14和21天的病情性状,接种后第7天所有株系均感病,病小穗率最低的为1.59%,第21天最高病小穗率为58.91%;孢子液喷雾接种后第21天材料全部感病,其中6棱株系的感病程度高于2棱株系。以病小穗率和病穗率划分赤霉病抗性的分布情况,发现病小穗率更能有效区分株系的赤霉病抗性。相关分析显示,病小穗率、病穗率、禾谷镰刀菌烯醇含量与粒色和穗密度呈显著负相关,而与株高、抽穗期无显著相关。     

表达Harpin蛋白质Hpa1功能片段的转基因小麦对赤霉病的抗性

来自水稻黄单胞菌的harpin蛋白质Hpa1有促进植物生长、诱导植物抗病性的功能,Hpa1序列10~42氨基酸片段（Hpa1_10-42）的活性比全长分子高1.3~7.5倍。为研究Hpa1_10-42在转基因小麦体内表达以后对赤霉病的影响和评价转基因小麦抗病水平与应用潜力,对6个小麦转基因系进行了测定。结果显示,Hpa1_10-42在转基因系T₃~T₅代呈现稳定表达,用禾谷镰刀菌一个分离物进行接种以后,转基因系发生赤霉病的程度较非转基因小麦显著降低,且转基因系T₃~T₅代赤霉病降低的趋势一致。另外,转基因系小麦病害减轻的程度,与在非转基因小麦上使用杀菌剂的效果相当,表明Hpa1_10-42转基因表达对小麦赤霉病有防治作用。     

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.     

哈尔滨地区抗瘟基因抗性分析及水稻品种抗性评价与利用

为明确哈尔滨地区24个抗瘟基因的抗性和20个水稻品种含有抗瘟基因型的情况,选取200个稻瘟病菌单孢菌株和22个已知无毒基因的鉴别菌株作为鉴定系统,通过喷雾法和离体划伤法接种,应用PCR技术对部分试验结果进行了重复性检测,并在此基础上进行了抗性布局分析。结果表明:24个抗瘟基因抗性差异较大,抗谱在11.00%~93.00%之间,平均抗谱38.29%;抗谱超过80.00%的基因有3个,由高到低为Pi-9、Pi-ta2和Pi-z5;抗谱低于20.00%的基因有9个,分别为Pi-a、Pi-ks、Pi-i、Pi-t、Pi-kp、Pi-ta、Pi-3、Pi-km和Pi-sh,Pi-a抗性最差。20个水稻品种中检测到抗瘟基因13个,共出现40次,其中Pi-a和Pi-19各出现6次,频率最高;龙稻5含抗瘟基因最多,4个,分别为Pi-ks、Pi-1、Pi-19和Pi-sh。在哈尔滨晚熟稻区建议布局绥粳8、中龙香粳1或者绥粳8+中龙香粳1搭配;在早熟稻区建议布局龙粳18、哈香07-321或者龙粳18+哈香07-321搭配。     

Biotransformation of clomazone in rice (Oryza sativa) and early watergrass (Echinochloa oryzoides)

Rice (Oryza sativa), a relatively tolerant species, and early watergrass (Echinochloa oryzoides; EWG), a relatively susceptible species, were exposed to ¹⁴C-labeled clomazone to determine accumulation, biotransformation, and mass balance. On a total mass basis, rice absorbed more clomazone than EWG (p < 0.05), but on a nmol/g basis, there was no significant difference between the two species (p > 0.05). Rice contained more extractable ¹⁴C residues (7.7 ± 0.5 vs. 4.8 ± 0.5 nmol in rice vs. EWG, respectively; p < 0.5), but the concentration in EWG was significantly higher (4.2 ± 0.5 vs. 1.8 ± 0.1 nmol/g in EWG vs. rice, respectively; p < 0.01). More metabolized residue was measured in EWG compared to rice (84.1% vs. 67.9%; p < 0.01). Both species produced hydroxylated forms, β-d-glucoside conjugates, and several other unidentified polar metabolites, but EWG generally produced higher metabolite concentrations. The concentration of the suspected active metabolite, 5-ketoclomazone, was significantly higher in EWG vs. rice (21 ± 2 vs. 5.7 ± 0.5 pmol/g, respectively; p < 0.01). Differences in sensitivity to clomazone between rice and EWG appear to be due to differential metabolism, but in this case the more susceptible EWG qualitatively and quantitatively metabolized more clomazone than the more tolerant rice. This is consistent with the action of a metabolically activated herbicide. This metabolic difference could be exploited to develop herbicide safeners for use with clomazone.