现代生物技术在棉花黄萎病菌分化研究中的应用

详细综述了营养体亲和群、同工酶、RFLP，PCR和RAPD等现代生物技术在棉花费萎病菌分化研究中的应用进展，并对基于分子标记技术的遗传分化与病菌致病性的关系进行了讨论。

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

Screening for Siderophore-producing Endophytic Bacteria against Fusarium oxysporum

[Objective] With a strain of endophytic antagonistic bacteria with good disease control efficacy in pot as trial material,the paper was to explore the antagonistic mechanism of siderophore against Fusarium oxysporum.[Method] Whether the strain produced siderophore and its fluorescent property was judged by MSA（Modified Sugar-Aspartic acid） plate.The siderophore activity of strains in liquid MSA medium was determined through the absorbance values at particular wavelength.The inhibition effects of it siderophore against F.oxysporum under different concentrations of Fe3＋ were compared.Then the strain was preliminarily identified based on morphological,physiological and biochemical characteristics [Result] The fluorescent siderophore produced by the endophytic bacteria in MSA medium had a strong inhibition effect against F.oxysporum.With the increasing concentration of Fe3＋,the inhibition effect against the pathogen weakened.The preliminary analysis showed these strains belonged to Bacillus.[Conclusion] Bacillus could compete the absorption of Fe3＋ by secreting siderophore to inhibit the growth of F.oxysporum.

A Preliminary Study on Biological Activity of Wikstroemia chamaedaphne Meissn

[ Objective ] The insecticidal and antibacterial bioactivity of Wikstroemia chamaedaphne Meissn were screened and bioactive substances in it were separated and purified. [ Method] The Wikstroemia chamaedaphne Meissn was conducted ultrasonic extraction in petroleum ether, ethyl acetate and methanol. The insecticidal activity of Wikstroemia chamaedaphne Meissn to Mythimna separata walker and aphid were determined. The antibacterial activity of Wikstroemia chamaedaphne Meissn to Fusarium graminearu, Glomerella cingulata, F. oxysporium f. sp niveum, Alternaria solani and Fusarium oxysporium were also determined. The bioactivity-guided methods such as opencolumn chromatography and Pre-HPLC method were used to separate active components in petroleum ether extract from Wikstroemia chamaedaphne Meissn. [ Result] When the concentration was 500 mg/L, 3 kinds of extracts from Wikstroemia chamaedaphne Meissn didn＇ t show obvious antibacterial bioactivity to 5 kinds of test samples. When the concentration was 5%, petroleum ether extract show certain topical toxicity to aphids. The ethyl acetate extract showed certain antifeedant activity to 3^rd instar Larvae of Mythimna separata Walker. The fraction F4 of petroleum ether extract possessed highest topical toxicity to aphids and the lethality was 60.00%. [ Conclusion] Wikstroemia chamaedaphne Meissn contained many insecticidal constituents whose active parts and mechanism were needed further researches.

Screening and Identification of Antagonistic Bacteria against Soil-borne Diseases of Tomato

[Objective] This study was conducted to screen resources for biological control of soil-borne diseases of tomato. [Method] Antagonistic bacteria against Ral- stonia solanacearum and Fusarium oxysporum were isolated and purified from soil samples by dilution plating and confronting incubation on PDA plates, then the antibacterial spectrum and metabolic secretions of the bacterial isolates were measured, and their species were identified by establishing phylogenetic trees with Neighbor-Joining method. [Result] From the 60 soil samples, 10 of 59 antagonistic bacteria isolates against R. solanacearum showed inhibition zone diameter 〉 25 mm, and 4 of 30 strains against F. oxysporum exhibited inhibition rate 〉 30%. The bacteriostatic substances and antibacterial spectrum analysis on above 14 isolates revealed that four strains N23-2, N58-2, NF59-3 and NF61-1 had good antago- nism against pathogenic bacteria and fungi; 12 strains produced cellulose, 11 strains produced proteases, and 6 strains produced siderophores. The molecular identification experiments indicated that four strains were members of Paenibacillus, three strains of Streptomyces, three strains of Pseudomonas strains, and four strains of Bacillus strains.

Inhibitive Activity of the Extracts of Plants in Artemisia against Fusarium oxysporum f. sp. vasinfectum and Fusarium momiliforme

[Objective]The aim was to provide theoretical basis for the control of cotton Fusarium wilt and cotton boll rot disease.[Method]The inhibitory activity of the extracts from three species of plants(Artemisia annual L.,Artemisia capillaris Thunb.,Artemisia argyi Levl.et Vant)against Fusarium oxysporum f.sp.vasinfectum and Fusarium moniliforme were studied under the condition of laboratory.[Result]The extracts of all the three plants in Artemisia showed strong antifungal activity against the tested pathogenic ...

Antagonistic Endophytic Bacteria Isolated from Different Growth Stages of Cotton for the Control of Cotton Verticillium wilt

[Objective] This study aimed to investigate the combined control effects of endophytic bacteria at different growth stages against cotton Verticfllium wilt and pro- vide a new strategy for the biocontrol of other soil-borne diseases. [Method] Endophytic bacteria with high resistance against Verticillium wilt were isolated from seedling, squaring and boll-setting cotton vascular, respectively. Their 16S rDNA se- quences were detected for comparative analysis. Three biocontrol strains were se- lected and identified, whose colonization roles in cotton plants were explored. The control efficiency was determined with indoor and field experiments. [Result] Accord- ing to the 16S rDNA sequence homology, the three strains were identified as Paeni- bacillus polyrnyxa YUPP-8, Paenibacillus xylanilyticus YUPP-1 and Bacillus subtilis YUPP-2, respectively. Results of colonization assessment showed that three strains all could be successfully colonized in cotton vascular. However, application amount had a positive effect on the number of colonized biocontrol bacteria in cotton, strain YUPP-8 had the largest number of colonized biocontrol bacteria in seedling period, strain YUPP-1 had the largest number of colonized biocontrol bacteria in squaring period, and strain YUPP-2 had the largest number of colonized biocontrol bacteria in boll-setting period. Indoor pot experiment showed that cotton plants in combined bio- control bacteria treatment group were not infected in flowing period, while Verticillium wilt morbidity rate of cotton treated with single strain in seedling period were 6.7% （YUPP-8）, 6.7% （YUPP-1） and 13.3% （YUPP-2）; however, Verticillium wilt morbidity rate wilt of the control reached 80%. Field experiment conducted during 2010-2011 showed that the combined application of three strains had better effect than separate application; specifically, Verticillium wilt morbidity rate and disease index of cotton in boll-setting period with combined application of three strains in 2010 were 9.4% and 6.5, respectively, while those in control group were 47.5% and 32.8; results in 2011 were similar to 2010, with higher disease severity. These results indicate that com- bined application of endophytic bacteria at different growth stages has great applica- tion potential in control of cotton Verticillium wilt. [Conclusion] This study preliminarily overcomes the defects in the application of biocontrol bacteria and provided reference for the prevention and treatment of other soil-borne diseases.

Effect of Chitinase-Producing Strain V-8 on Controlling Cotton Fusarium Wilt

[Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used as experimental materials to isolate endophytic bacteria. Through chitinase test and co-culturing both micro-or- ganisms side by side on the same PDA culture plate, antagonistic strains to cotton Fusarium wilt were screened. [Result] A total of 83 bacterial isolates were obtained from cotton plants grown in the fields, six of which were chitinase-productive bacte- ria. Through chitinase test and co-culturing both micro-organisms side by side on the same PDA culture plate, strain V-8 which had the strongest antagonistic effect on Fusarium oxysporum f. sp. vasinfectum was screened. Strain V-8 had a wider anti- fungal spectrum with certain inhibitory effect on all the six important pathogenic fungi including Fusarium oxysporum f. sp niveum; it colonized stably in the rhizospheric soil of cotton, with a colonization density of up to 6.2x10s cfu/g fifty days after inoc- ulation; the relative effect on controlling cotton Fusarium wilt in pot test was 73.2%. The Findings of this study suggested that strain V-8 had great potential for biological control of cotton Fusarium wilt and could be taken as a substantial material for the cloning of chitinase genes. [Conclusion] The results from this study provides bases for the control of cotton fusarium wilt, as well as the exploitation of endophytic bac- teria resources in cotton and the development of novel biological pesticides.

Effects of Medium Nutrients on Fusarium oxysporum Schl. f.sp

[Objective] To study the effects of different culture conditions on the Fusarium oxysporurn SchL f. sp. [Method] Based on species identification of the pathogenic organism of Fusarium oxysporum Schl. f. sp, effects of different cultures and different nutrients on the mycelial growth and conidial production of Fusarium oxysporum SchL f. sp were studied. [Result] The mycelial growth and conidial pro- duction of Fusarium oxysporum SchL f. sp was different under different culture con- ditions. PDA medium was the most suitable medium for the mycelial growth and had the highest conidial production; and the mycelial grew the fastest on the medium with maltose as carbon source or peptone as nitrogen source, which also had the highest conidial production. [Conclusion] This study provided experimental basis for the study of Fusarium oxysporum SchL f. sp and also provided theoretical basis for the study and control of Fusarium oxysporum Schl. f. sp.

Control Effects of Bacillus subtilis DJ-6 and Pyraclostrobin Alone and in Combination Against Fusarium oxysporum

[Objective] This study was conducted to screen a synergistic biological fungicide complex to control Fusarium wilt, reducing the use of chemical pesticides. [Method] The inhibitory effects of Bacil us subtilis DJ-6 and pyraclostrobin alone or in combination at five ratios against Fusarium oxysporum were detected by mea-suring mycelium growth rate in laboratory tests. The growth promotion and disease control effect of combined or single use of 20% pyraclostrobin and 2 ×1011 cfu/g B. subtilis DJ-6 WP at 1∶1 000, 1∶2 000 and 1∶3 000 dilutions were detected in field trials. [Result] The EC50 values of combined use of B. subtilis DJ-6 and pyra-clostrobin at ratios of 1∶1, 1∶2, 1∶3, 1∶4 and 1∶5 against F. oxysporum were 5.311 5, 4.008 6, 3.570 6, 3.350 9 and 3.218 9 μg/ml, with the synergistic ratios （SR） of 2.28, 1.77, 1.53, 1.64, 1.11, among which the synergetic effect at 1∶1 was the best. The fungicidal activity of pyraclostrobin was greater than that of B. subtilis DJ-6 in laboratory tests. Field trials revealed that al the 1∶1 000, 1∶2 000 and 1∶3 000 dilu-tions of 20% pyraclostrobin·2×1011 cfu/g B. subtilis DJ-6 WP in combination, 1∶1 000 dilution of 1 ×1012 cfu/g B. subtilis DJ-6 WP and 1∶2 000 dilution of 250 g/L pyra-clostrobin EC promoted the growth of strawberry by increasing plant height, leaf petiole, leaf blade area and stem diameter. Among them, the treatments with 1∶1 000 and 1∶2 000 of 20% pyraclostrobin · 2×1011 cfu/g B. subtilis DJ-6 WP in combina-tion had better effects than other treatments. The control effects of al the treat-ments were measured 30 and 80 d after fungicide application. The control effects of 1∶1 000 dilution of 20% pyraclostrobin and 2×1011 cfu/g B. subtilis DJ-6 in combina-tion were up to 100% and 93.11%, which were higher than those in al other treat-ments. The second highest control effects were found in the treatment with 1∶ 2 000 dilution of 20% pyraclostrobin and 2×1011 cfu/g B. subtilis DJ-6 in combination, they were 92.49% and 86.49%, higher than those in other treatments except the 1∶1 000 dilution of 20% pyraclostrobin and 2 ×1011 cfu/g B. subtilis DJ-6 in combination. The control effects of 1∶3 000 dilution of 20% pyraclostrobin and 2×1011 cfu/g B. subtilis DJ-6 in combination were 82.61% and 72.42%, higher than those in treatment with 1∶1 000 dilution of 1×1012 cfu/g B. subtilis DJ-6 WP, but lower than those in treat-ment with 1∶2 000 dilution of 25% pyraclostrobin EC. [Conclusion] Al the results re-vealed that the combination use of 20% pyraclostrobin and 2 ×1011 cfu/g B. subtilis DJ-6 WP at 1∶1 000 to 1∶2 000 dilution had better control effect against strawberry Fusarium wilt.

Preliminary Study on Garlic Root Exudates Influences to the Growth of Pomegranate Wilt Pathogen(Ceratocystis fimbriata)and Bacillus subtilis

[Objectives] This paper aims to explore the possibility to intercrop garlic with pomegranate tree to control pomegranate wilt.[Methods] Root exudates of garlic is cultivated in 1/5 concentration of MS solution and distilled water is examined in lab to test their effect to growth of mycelia of pomegranate wilt pathogen（Ceratocystis fimbriata）and multiplication of Bacillus subtilis.[Results] The result shows that garlic root exudates whatever cultivated in MS solution or distilled water could not inhibit or promote mycelia growth of C.fimbriata.However,garlic root exudates cultivated in both methods effectively promote multiplication of B.subtilis.[Conclusions] It is suggested that intercropping garlic with pomegranate tree by combining application B.subtilis could be a promising way to prevent pomegranate wilt spread in practice.

Purification of Antifungal Protein from Biocontrol Strain XG-1 against Fusarium Wilt of Watermelon

[Objective] This study aimed to isolate and purify the antifungal protein against Fusarium oxysporum f. sp niveum （FON） from Bacillus subtilis XG-I. [Method] The crude protein was obtained by using fractional precipitation with am- monium sulfate, then chromatography using both DEAE-Sepharose FF anion ex- change and Sephacryl S-100 gel filtration columns was adopted for further purifica- tion of the protein, followed by MALDI-TOF-MS analysis of the structure of the at- tained protein which was antagonistic to FON. [Result] The result from MALDI-TOF- MS analysis suggested that the antifungal protein extracted from strain XG-1 was highly homologous to the flagellin （gi114278900） deriving from B. subtilis, with a pro- tein score of 248 and a coverage rate of 63%. It was thus speculated that this an- tagonistic protein was a kind of flagellin with a molecular mass of 30.6 kD. [Conclusion] The results from this study provide theoretical basis for clarifying the mechanisms of the action of strain XG-1 as well as important references for the preven- tion and control of watermelon Fusarium wilt.

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L.

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O.sativa and O.meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.

Analysis of Rhizosphere Microbial Community Structure in Different Pathogenesis Stages of Watermelon Fusarium Wilt

High-throughput sequencing technique was applied to analyze the microbial community structure of rhizosphere soil at different stages of watermelon fusarium wilt to find out the difference of dominant microbial community in rhizosphere during the occurrence of watermelon fusarium wilt.Illumina-Hiseq high-throughput sequencing platform was used to sequence 16S and ITS rDNA in rhizosphere soil.The soil was named CK1 before planting,CK2 at peak stage and CK3 at stable stage.The results showed that the soil bacterial diversity was in the order of CK1>CK3>CK2,indicating no significant difference between CK1 and CK3 and a significant difference between CK2 and CK1,CK3.At the genus level,the dominant bacteria were Mizugakiibacter(21.9299%),Rhodanobacter(5.0933%),and Lactobacillus(3.1921%).The diversity of soil fungi was in the order of CK1>CK3>CK2,all showing significant differences.At the genus level,the dominant fungus was Lysurus(54.4601%),Papulaspora(12.4252%),Acrophialophora(3.1729%).The results showed that the diversity and abundance of bacteria and fungi in rhizosphere soil decreased during the peak period of watermelon fusarium wilt.With the gradual stabilization of the disease,the diversity and abundance of bacteria and fungi in rhizosphere soil recovered to a certain extent.

Gastric atrophy,diagnosing and staging

Hpylori is now accepted as the cause of gastritis and gastritis-associated diseases, such as duodenal ulcer, gastric ulcer, gastric carcinoma, and gastric MALT lymphoma. The natural history of H pylori gastritis includes inflammation progressing from the antrum into the adjacent corpus resulting in an atrophic front of advancing injury leading to a reduction in acid secretion and eventual loss of parietal cells and development of atrophy. Sub-typing intestinal metaplasia has no clinical value to the patient, the pathologist, or the endoscopist. The pattern, extent, and severity of atrophy, with or without intestinal metaplasia, is a far more important predictor than is intestinal metaplasia subtype. The challenge remains to identify a reliable marker that relates to pre-malignant potential.

Physiological Races of Fusarium oxysporum f. sp. Ciceris in Iraq

Twenty isolates of Fusarium oxysporum f. sp. ciceris were isolated from wilted chickpea plants obtained from different districts of north part of Iraq to assess variability in pathogenicity of the populations. Each isolate was tested on 12 differential chickpea varieties. Isolates showed highly significant variation in wilt severity on the differential varieties. Based on the reaction types that induced on differential varieties, isolates were grouped into four groups, First group included isolates FocSl, FocQ7, FocQ 10, FocFI3, FocH 17 and FocHl8; the second group included isolates FocS2, FocS3, FocS4, FocQ5, FocQ8, FocQ9, FocF11, FocF12, FocFl4 and FocH19; the third group included isolates FocF15, FocHl6, FocH20; where the isolate FocQ6 was placed in the fourth group. Results showed that the percentage of genetic similarity was ranged 42% to 100% and was 42% between the first group and other groups and 72% between the three groups the rest and thus this indicate the presence of four races of the fungus which are O, 4, 5 and 1B/C, this represent the first record of these races in lraq.

Comparison of methods of purification and identification of Verticillium dahliae toxins

The complexes excreted by VerticiUium dahliae are phytotoxins, which are responsible for most of the symptoms associated with Verticillium wilt disease. Verticillium dahliae toxins （VD-toxins） can be purified by different methods. In the present study, we reported a simpler, more effective method to purify VD-toxins. The supematant of V. dahliae culture was frozen, lyophilized and dialyzed by 1 kDa Dialysis Membranes （MWCO）. We also partially identified the characteristics of the purified VD-toxins. The results showed that the components of VD-toxins include glycoprotein within 35.8-83.2 kDa. The phytotoxic activity of VD-toxins was remained after VD-toxins were pretreated by high temperature, Concanavalin-A, and proteinase E, respectively. These data suggest that VD-toxins are heat-stable, and the protein fraction and glycosyl are both important contributors to the phytotoxic activity. VD-toxins purified effectively from the culture filtrates of V. dahliae may help in further understanding the mechanisms of interactions between V. dahliae and plants.

Use of Waste Vegetable Biomasses Treated by Steam Explosion for the Horticultural Crop Protection

The purpose of this study was to assess the suppressive effect of Waste Vegetable Biomasses （WVBs） treated by the Steam Explosion technique in a continuous plant, against soil-borne plant pathogens. In order to assess their disease suppression, five WVBs （Miscanthus biomass, durum wheat straw, rice straw, corn stalk and wood shaving） and commercial compost were tested in vivo at three different doses （10, 20 and 30% of potting mix） on seven horticultural pathosystems plant/fungus： tomato/Phytophthora nicotianae, cucumber/Pythium ultimum, lettuce/Fusariurn oxysporum f. sp. lactucae, melordFusariurn oxysporum f. sp. melonis, bearffRhizoctonia solani, eggplant/Verticillium dahlie and fennel/Sclerotinia sclerotiorum. The results showed that the corn stalk was more efficient respect to Miscanthus, compost, wheat straw, rice straw and wood shaving in all the patbosystems and at all the doses tested. The corn stalk suppression ranged from 97% in eggplant/F, dahliae to 35% in lettuce/F, oxysporum f. sp. lactucae, and it was significantly higher with respect to the other substrates. In general, the wheat straw, rice straw and wood shaving were statistically found less efficient as suppressive substrate with respect to corn stalk, Miscanthus and compost at the 30% dose in four pathosystems In particular, the wood shaving suppressiveness ranged from 48% in eggplant/V, dahliae to 12% in lettuce/F, oxysporum f. sp. lactucae. The different suppressiveness observed could be attributed to different concentration of the microbial inhibitory substances （furfurals, organic acids and lignosulfonates） produced during the processing of fresh biomass.

Controlling of Fusarium Wilt of Cucumber by Antagonistic Bacteria

Studying about the effect of four pseudomonas and two serratia isolates on growth of Fusarium oxysporum showed that, Pseudomonasfluorescens No.2 ＆ No.3 and Serratia marcescens No.2 gave highest inhibition zones which were 37.33, 35,00 and 31.33 mm, respectively. Evaluating about the effect ofpseudomonas and serratia culture filtrates at three concentrations （10, 25, 50%） on the linear growth and spore germination ofFusarium oxysporum revealed that, all filtrates of the tested isolates reduced the mycelial growth and spore germination of F. oxysporum. All filtrates of the tested isolates at 50% concentration completely inhabited spore germination ofF. oxysporurn. Culture filtrates of Pseudomonasputida and Serratia marcensens No.2 at 50% concentration reduced the mycelial growth of F. oxysporurn by 80.74 and 80.37% respectively. In this respect, all tested isolates made lysis to mycelial of F. oxysporum except Pseudornonasfluorescens No.3. Effect of treating cucumber seeds with cell suspension of pseudomonas and serratia isolates on incidence of Fusarium wilt disease revealed that all isolates were effective in reducing disease incidence and disease severity and increasing the percentage of healthy plants compared to the control. Pseudornonasfluorescens No.3 and Serratia marcensens No.2 were the best isolates and completely prevented the disease incidence.

Isolation and Identification of Main Fungal Pathogens in Muskmelon in Open Field of Hubei Province in Summer

Fungal diseases often occur seriously in muskmelon in open field of Hubei Province in summer, especially in continuous cropping pattern, resulting in great economic losses. In this study, the pathogens of main fungal diseases in muskmelon in open field of Hubei Province were isolated, and they were identified by morphological and molecular techniques. The results showed that muskmelon fusarium wilt is a major disease in muskmelon in open field of Hubei Province in summer, and its pathogen was confirmed to be Fusarium oxysporum. In future studies, one pair of specific primers would be designed to detect different pathogenic races of Fusarium oxysporum so as to accelerate the detection and to shorten the detection time,thereby proving guidance for actual production.