丹皮根石油醚萃取物对水稻纹枯菌的毒力评估及化学成分分析

水稻纹枯病由水稻纹枯菌引起,是危害水稻最严重的病害之一。虽然化学杀菌剂是主要的控制方法,但其长期不合理的施用导致产生环境污染、食品风险、非靶标生物中毒等问题。因此,低毒的植物源杀菌剂成为理想的替代品,引起了研究者的关注。在前期研究中发现,丹皮根乙醇粗提物在1000μg·mL-1时,对水稻纹枯菌有优异的抑菌活性,抑菌率为100%。进一步研究发现,抑菌活性物质主要集中在石油醚萃取物中,但石油醚萃取物对水稻纹枯菌的毒力大小及化学成分还不清楚。本研究在评估丹皮根石油醚萃取物对水稻纹枯菌毒力大小的基础上,采取GC-MS分析丹皮根石油醚萃取物的化学成分种类及含量。结果表明,石油醚萃取物的提取率为1.17%,对水稻纹枯菌的有效抑菌中浓度EC50值58.65μg·mL-1,最低抑菌浓度为120μg·mL-1。石油醚萃取物的成分有4个,为丹皮酚、棕榈酸、亚油酸和β-谷甾醇,且丹皮酚的含量超过97%,为最主要成分;其次为β-谷甾醇,含量超过1.8%;棕榈酸、亚油酸的含量较少,均低于1%。本研究表明,丹皮根石油醚萃取物对水稻纹枯菌具有较好的抑菌活性,化学成分分析为其开发为水稻纹枯菌生物杀菌剂提供了理论支撑。

水稻纹枯菌总DNA提取方法的比较

DNA的浓度,尤其是DNA的纯度严重影响后续基因组酶切等结果。为寻找一种高质量的水稻纹枯菌总DNA的提取方法,采用CTAB法、CTAB改良法、SDS法、蜗牛酶法、蛋白酶K法5种方法分别提取水稻纹枯菌总DNA。通过紫外吸光度测定DNA的纯度和浓度、琼脂糖凝胶电泳、PCR扩增、限制性内切酶酶切反应及Southern杂交评价DNA的质量,最终得出蛋白酶K法是获取高质量水稻纹枯菌基因组DNA的最佳方法。

脉冲电场对水稻纹枯菌的杀灭效果

探讨了脉冲电场杀菌的机理,仿真分析了培养液中脉冲电场的分布情况,仿真结果表明:进入培养液内脉冲电场时域波形的脉宽都有不同程度的变窄;脉冲上升沿越陡峭,进入培养液内部的脉冲电场峰值越高,穿透性越好。对脉冲电场水稻纹枯菌杀灭效果进行了试验研究,在同轴型处理室中分别以不同幅值的电磁脉冲(频率为2.5Hz)和不同的作用时间对10-4稀释的水稻纹枯菌菌液进行处理,试验结果表明:当电磁脉冲的幅值较低时,可以通过延长灭菌的时间来提高灭菌效果;而幅值较高时,则脉冲幅值为灭菌效果的主要影响因素,延长灭菌时间的意义不大。

防治水稻纹枯病菌的室内药剂筛选

采用菌丝生长速率法,用6种常见的杀菌剂对水稻纹枯菌进行室内毒力测定。结果发现,醚菌酯和苯醚甲环唑的抑菌效果比较好,EC50分别为0.270 1 mg/L和0.766 2 mg/L;其次为菌核净、多菌灵和三唑酮;抑菌效果最差的是福美双,其EC50值达到5.392 5 mg/L。

山苍子花蕾挥发油的提取、鉴定及抗植物病原菌活性研究

通过水蒸气蒸馏法从山苍子花蕾中提取山苍子花蕾挥发油,利用GC-MS仪对其化学成分进行分析,并采用菌丝生长速率法测试山苍子花蕾挥发油对水稻纹枯、苹果腐烂、辣椒疫霉、番茄灰霉、马铃薯晚疫和小麦茎基腐6种植物病原菌的抑菌活性;通过扫描电镜观察了山苍子花蕾挥发油对水稻纹枯病原菌超微结构的影响,并分析水稻纹枯病原菌的生理学变化。结果表明,山苍子花蕾挥发油中含有24种有效成分,其中反式柠檬醛含量为6.00%,桉叶油醇为6.13%,顺式柠檬醛为7.00%,柠檬烯含量高达38.85%;山苍子花蕾挥发油对水稻纹枯病原菌具有较好的抑制效果,其半数有效浓度(IC_(50)值)为150.138μg/mL;相较于空白组,山苍子花蕾挥发油处理水稻纹枯病原菌5 d后,菌丝的鲜重和干重分别降低74.36%和68.73%,MDA含量上升50.42%;在IC_(50)浓度下,菌丝的电导率最终上升75.34%。说明山苍子花蕾挥发油对菌丝细胞膜具有一定的破坏作用,导致菌丝体内电解质大量外泄,难以维持菌丝正常的生理生化功能,并导致了菌丝的死亡。以上结果为山苍子的高值化利用提供一定的理论参考。

酿酒下脚料在水稻病害防治中的应用探究

酿酒下脚料是白酒固态酿造过程中的副产物,富含有机酸类化学物质。对于酿酒下脚料的处理,绝大多数酒厂都是直接丢弃,这样既造成了浪费,更造成了严重的环境污染。通过对水稻稻瘟菌和水稻纹枯菌的敏感性实验发现,酿酒下脚料对两者菌丝的生长具有明显的抑制作用。

菊芋叶片提取物抑菌活性与化学成分的研究

为了开发新型植物源杀菌剂和充分利用菊芋(Helianthus tuberosus L.)资源,本文尝试用石油醚、乙醚、乙酸乙酯和水等4种溶剂对菊芋叶片进行平行提取,采用生长速率法测定菊芋叶片提取物对水稻纹枯菌(Rhizoc-tonia solaniKühn)、小麦赤霉菌[Gibberella zeae(Schw.)Petch]、番茄早疫菌[Alternaria solani(Ellis et Martin)Jones et Grour]和番茄灰霉菌(Botrytis cinerea Pers.)生长量的抑制活性;并通过试管法和滤纸法对菊芋叶片内化学成分进行初步预试。抑菌实验结果表明:(1)菊芋叶片各溶剂提取物处理与对照处理相比差异显著;(2)菊芋叶片水提取物处理与各有机溶剂提取物处理差异也基本显著;(3)菊芋叶片各溶剂提取物同浓度处理对水稻纹枯菌、番茄早疫菌和番茄灰霉菌抑制效果较好;(4)菊芋叶片乙酸乙酯提取物抑菌效果最为显著,浓度为20mg/mL时对番茄早疫菌和番茄灰霉菌已达到完全抑制,对水稻纹枯菌抑制率也达到77.91%。初步化学预试结果说明,菊芋叶片中含有蛋白质、氨基酸、还原糖类、有机酸、酚类和鞣质、黄酮类、内酯类、强心甙以及油脂等化学成分。

Cloning and Analysis of WRKY Gene of Rice Induced by Rhizoctonia solani Kuhn

[Objective] The aim was to clone the up-regulated expression gene of rice induced by Rhizoctonia solani.[Method] The EST fragment K16 obtained by suppression subtraction hybridization（SSH）was cloned and confirmed by reverse transcription-polymerase chain reaction（RT-PCR）.Then RT-PCR products were cloned into the PMD18-T vector and sequenced.The functions of the sequence were predicted with bioinformatics method.[Result] A 1 079 bp gene was obtained.The gene encoded a protein with 236 amino acids.The protein contains many motif sites,two WRKY domains and a C2H2 zinc finger motif.The gene showed high identities with WRKY8,WRKY24 and WRKY30 gene of rice.[Conclusion] The up-regulated expression gene induced by R.solani was representative WRKY family gene.The gene could play an important role on rice sheath blight resistance.

Preliminary Study on the Extraction of Crude Toxin of Rhizoctonia solani and Its Activity

[ Objective] This study was to investigate the pathogenic mechanism of rice sheath blight pathogen （ Rhizoctonia solani） and the bioactive components of toxin. [ Method ] Rice sheath blight pathogen was cultured in the improved Richared medium; the culture filtrate was centrifuged and sterilized, then treated by activated carbon adsorption chromatography, distilled with methanol or water, and all were next concentrated, yielding the crude extracts of culture solution, crude extracts of methanol and crude extracts of water; the activities of these three extracts were determined, [ Result] The three extracts were russet pastes; activity determination showed that they had remarkable inhibitory effects on the growth of rice radicle and plantule, as well as the growth of four-foliage-young seedlings. They could also generate toxic effects on abscisic foliages and spots similar to the symptoms of sheath blight pathogen. [ Conclusion] Bioactive components of rice sheath blight pathogen toxin may be composed of various ingredients.

Effect of Morphological Traits on Sheath Blight Resistance in Rice

Sheath blight, caused by the Rhizoctonia solani Kuhn, is one of the most serious diseases of rice (Oryza sativa L.). The relationship between morphological traits and sheath blight resistance was investigated in 1999 and 2000 by using a segregant population consisting of 240 inbred recombination lines, derived from an elite combination of Zhenshan 97 X Minghui 63. Partial correlation analysis between phenotypic data (morphological characteristics and heading date (HD)) and sheath blight ratings (SBR) showed that only one trait, plant compactness, was significantly correlated with resistance to sheath blight in both 1999 and 2000, eight traits, such as plant height, heading date, and penultimate leaf angle, were significantly correlated with SBR in either of the two years, and the remaining 10 morphological traits were not consistently associated with SBR in the two years. A major quantitative trait locus (QTL) controlling plant compactness was found to be in the site on chromosome 9 adjacent to the region responsible for qSB9, a major QTLs conferring sheath blight resistance. One out of the three QTLs contributing to a penultimate leaf angle was mapped approximately on the same region as another sheath blight resistance QTL, qSB5, located on chromosome 5. Whereas, no QTLs underlying most other traits was detected on the chromosomal region correlated with sheath blight resistance QTL. The results in the present study suggested that the morphological traits were not the main factors responsible for the SBR separation among the recombinant inbred lines (RILs) population. Nevertheless, some morphological characteristics had some indirect influence on expression of sheath blight resistance genes through altering a microclimate on paddy field so as to influence the infection of the pathogen and development of the disease. An efficient approach in resistance breeding to sheath blight was recommended by pyramiding major QTLs for sheath blight resistance and selecting those morphological traits that favor resistance gene expression.

Antifungal Effect of Streptomyces 702 Antifungal Monomer Component DZP8 on Rhizoctonia solani and Magnaporthe grisea

The aim of this study was to investigate the in vitro antifungal effects of antifungal monomer component DZP8 isolated from Streptomyces 702 on the mycelium growth, sclerotium formation and germination of Rhizoctonia solani and on the mycelium growth, conidial formation, germination, appressorium formation of Magnaporthe grisea. The results showed that the antifungal monomer component DZP8 has strong antifungal effect on both the R. solani and M. grisea. The EC50 and EC90 of DZP8 were 1.81 and 3.35 μg/ml on Ft. solani respectively, and 37.01 and 136.21 μg/ml on M. grisea respectively. Under the treatment of 48.01 μg/ml DZP8, the sclerotium formation rate of R. solani was just 39.21%, the formation time delayed by 216 h and the dry weight decreased by 81.37% in comparison the con- trol; and 33.51 μg/ml DZP8 significantly inhibited the sclerotium germination. In the presence of 160.08 μg/ml DZP8, the sporulation of M. grisea was just 9.29% of control sample; 20.14 μg/ml DZP8 inhibited the conidial germination suppression rate by 95.16%, and the appressorium formation by 100%.

The Effect of Three New Fungicides against Rice Sheath Blight in Field Experiment

[Objective] The aim was to study the effect of three new fungicides against rice sheath blight in field experiment. [Methods] The experiment set up 7 treatments with three times of repetition and designed by random grouping. By using 5 sampling points in each plot, and investigating continuous 4 holes of each point, total plants, diseased plants and disease degrees were recorded. Then disease index and control efficiency were calculated, and variance analysis was carried out. [Results] 300 or 450 ml/hm^2 azoxystrobin ＋ difenoconazole 325 g/L SC had better control efficiency to rice sheath blight and had no phytotoxicity effect, we should use it at the initial disease stage and continuously spray 2-3 times. [Conclusion] The experiment provided a theoretical basis for controlling rice sheath blight using fungicides.

Mapping QTLs Associated with Sheath Blight Resistance Using Chromosome Segment Substitution Lines of Rice(Oryza sativa L.)

In this study, a population of 119 chromosome segment substitution lines （CSSLs） derived from backcross between indica 9311 and japonica Nipponbare was employed to map quantitative trait loci （QTL） associated with sheath blight resis-tance in rice with toothpick inoculation method. A total of three sheath blight resis-tance-associated QTLs （qsb8-1, qsb8-2 and qsb8-3） were identified, which were lo-cated on adjacent molecular markers RM3262, RM5485 and RM3496 of chromo-some 8; the genetic interval was 81.7cM-91.7cM, 91.7cM-108.1cM and 108.1cM-119.6cM, respectively. The additive effect of qsb8-2 was negative, indicating that sheath blight resistance of susceptible parent harboring qsb8-2 fragment was en-hanced; additive effects of qsb8-1 and qsb8-3 were positive, indicating that sheath blight resistance of susceptible parent harboring qsb8-1 and qsb8-3 fragments was reduced.

Control Efficacy of Validamycin A·(0.2 Billion Spores/ml) Paenibacillus polymyxa DN-1 3% AS for Rice Sheath Blight

With validamycin A.（0.2 billion spores/ml） Paenibacillus polymyxa DN-1 3% AS as the test agent, the effects of different dosage and different application time on the control efficacy for＇ rich sheath blight were investigated. The results of two- year test showed that when the application amount was in the range of 45-90 g.a.i/ hm^2, the field efficacy of validamycin A-（0.2 billion spores/ml） P. polymyxa DN-1 3% AS in the initial infection stage of rich sheath blight （Le., the diseased plant rate was below 5%） reached 80.38%-89.06%, and that in the peak infection stage （i.e., the diseased plant rate was higher than 10%） reached only 41.12%-53.26%. The field efficacy of validamycin A.（0.2 billion spores/ml） P. polymyxa DN-1 3% AS at the early onset of rich sheath blight was significantly better than that at the onset, so that the application time of validamycin A .（0.2 billion spores/ml） P. polymyxa DN-1 3% AS should be appropriately brought forward in the prevention and control of rice sheath blight.

Screening of Rice Stripe Virus(RSV)-resistant Germplasm from Landraces of Rice(Oryza sativa L.)

[Objective] The aim of this study was to screen rice strip virus （RSV）-resistant landraces. [Method] The resistance of 119 rice landraces to rice stripe virus was identified in field spontaneously infected with smal plant-hopper. [Result] There were 55 landraces resistant to rice strip disease in 56 indica rice landraces, but on-ly two resistant to rice strip disease in 63 japonica rice landraces. [Conclusion] The results revealed that there were abundant rice landscapes resistant to RSV in Chi-na, and these varieties can be used to develop more genes resistant to RSV.

Mapping of QTLs for Sheath Blight Resistance Using Recombinant Inbred Lines of Rice(Oryza sativa L.)

A recombinant inbred line （RIL） population composed of 157 lines derived from an inter-subspecific hybrid of Daguandao/IR28 by the single seed descent method was used as materials, and the quantitative trait loci （QTLs） coffering the resistance to sheath blight in the 157 RILs and the parents were detected using the toothpick inoculation method. The disease indexes of rice sheath blight in the two parents and 157 RILs were scored and the QTLs responsible for rice sheath blight resistance were detected accordingly by QTL Cartographer software. The results showed that a total of 4 QTLs （qsbl, qsb2, qsb5-1, qsb5-2） conferring sheath blight resistance were detected on chromosomes 1, 2 and 5, and their variance explained ranged from 10.41% to 36.92%. The additive effect of qsb5-1 was negative, indicat- ing that the QTLs derived from donor parent IR 28 could enhance the resistance to sheath blight. However, the additive effects of qsbl, qsb2 and qsb5-2 were positive, indicating that the QTLs derived from donor parent Daguandao weakened the resis- tance to sheath blight.

Study on the control of rice stripe disease with Bilken virusicide

The paper reviewed the function mechanism of Bilken virusicide against rice stipe disease, and then introduced its control effects in field test as well as its application method.

Efficacy of Difenoconazole-azoxystrobin 32.5% SC in Treatment of Rice Sheath Blight and Its Application Technology

[Objective] The efficacy of difenoconazole-azoxystrobin 32.5% SC for rice sheath blight and its application technology were discussed in this research. [Method] Three surveys were carried out. There were 5 fixed test clumps in each plot. The number of total plants and disease plants and disease progression in each fixed clump were recorded. The correlation effectiveness was calculated based on the growth rate of disease index. Significance analysis was performed with Dun- can＇s new multiple range method （DMRT）. [Result] The difenoconazole-azoxystrobin 32.5% SC had a good efficacy for rice sheath blight, and its efficacy increased with the increase of dose. If sprayed according to the dose of 450 ml/hm2 5 days before the beginning of heading stage of rice, difenoconazole-azoxystrobin 32.5% SC would have better effects on controlling rice sheath blight with correlation effectiveness reaching up to 95.14%, which was 2.06% higher than that of control drug （trifloxys- trobin-tebuconazole 75% WG, 225 g/hm2）. [Conclusion] Treated with difenoconazole- azoxystrobin 32.5% SC with dose of 450 ml/hm2, rice would have green upper leaves and less yellow middle and lower leaves. Moreover, the yield was in- creased significantly. The difenoconazole-azoxystrobin 32.5% SC had good safety. Therefore, difenoconazole-azoxystrobin 32.5% SC had a good application prospect in production.

A Resistance Bioassay for Rhizoctonia Root and Crown Rot and Damping-off Caused by the Anastomosis Groups AG 2-2111B and AG 4 in Sugar Beet

In order to determine the level of resistance of sugar beet varieties against Rhizoctonia solani AG 2-21IIB and AG 4, a methodology was implemented under greenhouse conditions that contemplated the most important criteria regarding to plant-pathogen interaction. The effect of plant growth stage on the development of the disease was evaluated. Seven sugar beet varieties were tested for resistance to R. solani AG 2-2IIIB and AG 4. To detect differences in leaf temperature between/L solani inoculated plants and non-infected plants, an infrared （IR） camera was tested. High incidence of R. solani AG 2-2IIIB and AG 4 in sugar beet plants was evident when the fungal inoculum was applied to two and four weeks old plants. At four weeks after sowing, it was the optimum time to inoculate sugar beet plants in order to generate R. solani infection, since at this time all plants were infected. Significant differences were detected regarding disease incidence between sugar beet varieties inoculated with different anastomosis groups. Leaf temperature was significant different between inoculated and non-inoculated plants, demonstrated that this technique could be a new tool for breeders to screen for resistance of new varieties.

Effect of Soil Amendments on Soil Borne Pathogens of French Bean （Phaseolus vulgaris L,） in Organic Farming System in Manipur

The rationale behind the use of soil amendments by traditional marginal farmers against soil borne pathogen is an area of interest. The present work ascertain the effect of certain soil amendments against pre emergent and post emergent rot of French bean in the agro climatic condition of Manipur, Four soil amendments： cow dung （COD）, sawdust （SD） ＋ paddy husk （PH）, decomposed floating phytomat （DFP） was tried in field conditions in four different combinations. These have been often used by the traditional farmers. A locally available commonly cultivated pole type of French bean has been used for the study. Disease incidences in plots treated with these amendments were then compared with that of control plots where no amendments were made. The plots with a combined treatment of the soil amendments showed maximum seed germination and least post emergent rot against sclerotinia rot. But DFP showed effectiveness against rhizoctonia rot with maximum seed germination while saw dust showed maximum decrease in post emergent rot against rhizoctonia rot. The plots treated with organic soil amendments showed statistically significant positive effect against the pathogens as compared with untreated control plots.