Biological Characteristics of Fusarium oxysporum f. sp.niveum Isolated from Watermelon Cultivated in Plastic Greenhouse and Indoor Toxicity Determination of Different Fungicides in Hainan Province of China

[ Objective] The paper was to study biological characteristics of Fusarium oxysporum f. sp. niveum in watermelon cultivated in plastic greenhouse in Hainan Province of China and to determine indoor toxicity of fungicides. [ Method] Effects of various factors on mycelial growth and spore germination were stud- ied, and the lethal temperature of spores and indoor toxicity of fungicides were also determined. [Result] The fungal mycelium had a better growth rate on water- melon juice medium than the others. The optimum temperature for mycelial growth and spore germination were 28 ℃ and 30 ℃, and the optimum pH values were 7 - 8 and 7 - 9, respectively. Light-dark alternation was conducive for mycelial growth. The lethal temperature of spore was 60 ℃ for 5 min. The fungal mycelium grew better while taking fructose and glucose as carbon sources and yeast extract and peptone as nitrogen sources. Toxicity determination showed that 50% pro- chloraz-manganese chloride complex WP had the greatest inhibition effect on mycelial growth, with the ECso value of 0.730 9 μg/mL; followed by 32.5% benzoic azoxystrobin SC, 30% benzoylate· propieanazol EC, 10% difenoconazole WG, 25% bromothalonil · carbendazim WP and 25% bromothalonil WP, with the ECso values from 1.884 7μg/mL to 8. 161 0μg/mL. [Conclusion] The study provided basic data for field control against F. oxysporum f. sp. niveum on watermelon cultivated in plastic greenhouse in Hainan Province.

DNA methylation patterns of banana leaves in response to Fusarium oxysporum f. sp. cubense tropical race 4

Fusarium wilt of banana, which is caused by Fusarium oxysporum f. sp. cubense tropical race 4 （Foc TR4）, is a serious soil-borne fungal disease. Now, the epigenetic molecular pathogenic basis is elusive. In this study, with methylation-sensitive amplification polymorphism （MSAP） technique, DNA methylation was compared between the leaves inoculated with Foc TR4 and the mock-inoculated leaves at different pathogenic stages. With 25 pairs of primers, 1 144 and 1 255 fragments were amplified from the infected and mock-inoculated leaves, respectively. DNA methylation was both changed and the average methylated CCGG sequences were 34.81 and 29.26% for the infected and the mock-inoculated leaves. And DNA hypermethylation and hypomethylation were induced by pathogen infection during all pathogenic stages. Further, 69 polymorphic fragments were sequenced and 29 of them showed sequence similarity to genes with known functions. And RT-PCR results of four genes indicated that their expression patterns were consistent with their methylation patterns. Our results suggest that DNA methylation plays important roles in pathogenic response to Foc TR4 for banana.

5-Aminolevulinic acid against strawberry Fusarium wilt:Bidirectional regulation of biocontrol agents and pathogens

Strawberry Fusarium wilt (SFW) is a systematic soil-borne disease caused by Fusarium oxysporum f.sp.fragaria (Fof),which infects the vascular bundles,blocking water and nutrient transport from roots to the aboveground.It is a severe pathogen which spreads rapidly and destroys strawberry production.Finding a way to control this disease is of great scientific value and practical importance.In this study,three fungi were isolated from the vascular tissues of sick strawberries in the field.After DNA sequencing,they were identified as Fof,Aspergillus fumigatus and Trichoderma harzianum,respectively,among which the first two are pathogens and the third is a probiotic.All fungi were controlled by thiophanate-methyl (TM),a commercial fungicide.On PDA medium,20 mg·L^(-1)5-aminolevulinic acid (ALA),a natural non-protein amino acid,promoted T.harzianum proliferation,but inhibited Fof and A.fumigatus.In confrontation test,the growth of Fof or A.fumigatus was inhibited by T.harzianum and exogenous ALA promoted T.harzianum growth but significantly inhibited the pathogen growth.When three species of fungi were separately or combinedly inoculated on healthy strawberry plants,T.harzianum promoted plant growth and development while Fof or A.fumigatus caused growth retardation,where Fof directly caused leaf yellowing and plant wilting.When the plants inoculated with different fungus were treated with ALA,the results turned out that ALA alleviated SFW symptoms by bidirectionally promoting T.harzianum proliferation and inhibiting Fof and A.fumigatus.Thus,ALA might be used in comprehensively controlling SFW in strawberry industry.

河北省西瓜枯萎病菌生理小种鉴定与AFLP分析

【目的】明确河北省西瓜枯萎病菌生理小种类型和遗传多样性,为西瓜抗病育种和西瓜枯萎病的综合防治提供依据。【方法】本文对采自河北省12个西瓜种植地区的46个西瓜枯萎病菌菌株和4个已知生理小种的菌株进行了致病性测定和AFLP分析。【结果】根据菌株对3个鉴别寄主表现出的致病性强弱,将河北省46个西瓜枯萎病菌菌株划分为3个不同的生理小种,即0号、1号和2号,分别占供试菌株的17.4%、65.2%和17.4%,其中1号生理小种为优势小种,分布在河北省所有西瓜种植区。21对AFLP引物对供试菌株扩增出1157条带,其中多态性带389条,占总带数的33.6%。河北省西瓜枯萎病菌的遗传距离变化在0.33～0.95之间,平均为0.66,群体遗传多样性比较丰富。基于AFLP标记聚类分析表明,50个菌株被划分为3个类群(AFLP Groups,AGs)。AGI包含4个菌株,均为2号生理小种;AGII包括4个菌株,均为0号生理小种;AGIII包括42个菌株,以1号生理小种为主(32个),占该类群的76.2%。【结论】河北省西瓜枯萎病菌存在明显的致病性分化,其AFLP类群划分与致病性鉴定划分的生理小种之间存在一定相关性,但与菌株的地理来源无关。

GFP标记的尖孢镰刀菌西瓜专化型侵染西瓜过程观察

将绿色荧光蛋白基因转入西瓜尖孢镰刀菌FON中,并利用荧光共聚焦显微镜观察GFP标记菌株侵染西瓜的过程。结果显示,转化子连续转接4代能够稳定遗传,荧光强度良好,PCR验证gfp基因已转入菌株FON中;利用GFP标记菌株在荧光共聚焦显微镜下观察其侵染西瓜苗的过程,发现在1/2 MS培养的西瓜苗中,FON经过48 h侵染即可进入西瓜的根维管束,第3天便进入茎维管束,第4天进入叶维管束(包括叶柄和叶脉);在土壤盆栽条件下,侵染后2 d FON进入西瓜的根维管束,第9天进入茎维管束,第11天进入叶维管束。培养基培养与土壤盆栽相比,培养基栽培FON侵染的速度更快。

Arbuscular Mycorrhizal Fungal Inoculation Enhances Suppression of Cucumber Fusarium Wilt in Greenhouse Soils

A pot experiment was conducted to study the plant growth and fruit yields of cucumber （Cucumis sativus L.） on a greenhouse soil with or without inoculation of arbuscular mycorrhizal fungi （AMFs） and Fusarium oxysporum f. sp, cucumerinum under unsterilized conditions. Two AMF inocula were tested： only one AMF strain Glomus caledonium 90036 and an AMF consortium mainly consisting of Glornus spp. and Acaulospora spp. There were four treatments including no inoculation （control）, inoculation with F. oxysporum but without mycorrhizae （FO）, inoculation with F. oxysporum and G. caledonium （FO＋M1）, and inoculation with F. oxysporum and the AMF consortium （FO＋M2）. Cucumber plants were harvested at weeks 3 and 9 after transplanting. Compared with the control, the FO treatment without AMF inoculation had less biomass both at weeks 3 and 9 （P 〈 0.05） and had higher incidence of Fusarium wilt and produced no cucumber fruit at week 9. Both FO＋M1 and FO＋M2 treatments had higher mycorrhizal colonization than the treatments which received no AMF inoculation at week 3 （P 〈 0.05）, but only the FO＋M2 treatment elevated plant biomass, decreased the incidence of Fusarium wilt, and improved cucumber yields to the same level as the control at week 9. The results indicated that the AMF consortium could suppress Fusarium wilt of cucumber and, therefore, showed potential as a biological control agent in greenhouse agroecosystems.

根际施用微生物有机肥防治连作西瓜枯萎病研究

为了探讨西瓜专用微生物有机肥料（BOF）对西瓜植株枯萎病的防治效果，采用盆栽试验研究了西瓜专用BOF对连作土壤上西瓜植株生长和枯萎病的防治效果以及西瓜枯萎病致病菌的数量分布的影响。结果表明：1）营养钵育苗和移栽土壤中都施用BOF的处理，未发现西瓜枯萎病植株，而对照植株则完全发病；2）施用BOF处理的致病菌（尖孢镰刀菌西瓜专化型）数量在根际土壤中为0.7×103 cfu/g，在土体土壤中为2.7×103 cfu/g，都控制在104 cfu/g数量级以下，而对照处理的数量分别达到了1.17×105 cfu/g和1.1×105 cfu/g；3）与对照比较，营养钵育苗时施用1%的BOF，西瓜苗期（播种后17 d）生物量都显著增加，根系生物量比地上部生物量提高了16.7个百分点（以鲜重计）和24.8个百分点（以干重计）；营养钵育苗和移栽土壤中均施用BOF的处理，西瓜植株（生长67 d）生物量（干重）是对照的1.83倍；在营养钵育苗或移栽时施用BOF的处理，西瓜植株干重两者差异不大，分别是对照的1.28倍和1.27倍。采用营养钵育苗和移栽时都施用BOF的方法种植西瓜，能有效地促进西瓜植株生长，防止西瓜枯萎病发生，克服西瓜连作障碍。

西瓜不同品种苗期感染枯萎病菌后的生理生化变化

研究了西瓜不同品种苗期感染枯萎病菌 (FusariumoxysporumSchlecht.f.sp .niveum)后根部细胞的质膜透性、某些酶类和化合物 ,以及光合色素含量的动态变化。结果表明 ,受枯萎病菌侵染后 ,抗病品种克伦生在染病后的短时间内 (48h左右 )能使细胞内MDA、相对电导率、SOD酶和CAT酶的代谢基本恢复到正常状态 ,其自我调节恢复正常状态的能力显著大于感病品种早花 ;抗病品种比感病品种具有保持较高脱氢酶活性的能力 ;抗病品种Vc含量明显高于感病品种 ;抗病品种可溶性糖的相对含量要比感病品种低 ;接菌 1d后抗病品种可溶性蛋白含量高于感病品种 ;抗病品种抑制叶绿素含量下降的能力和保持较高类胡萝卜素含量的能力明显强于感病品种。

香蕉抗(耐)枯萎病新品种桂蕉9号的选育及其高产栽培技术

【目的】选育抗（耐）尖孢镰刀菌古巴专化型4号生理小种（FOC4）引起的香蕉枯萎病,且产量、品质性状优良的香蕉新品种,丰富广西抗（耐）枯萎病香蕉品种,为促进广西香蕉产业的健康持续发展提供技术支持。【方法】采用重病蕉区大田筛选母株,并利用组织培养芽变、盆栽接种病原菌压力选择及大田病区压力选择相结合的方法选育抗（耐）香蕉枯萎病品种,经过品系纯化、品比试验、区域试验、生产试验等,观察其特征特性、抗病性及产量表现等。【结果】桂蕉9号为巴西蕉芽变异株系,基因型为AAA,属中秆香蕉,全生育期310～350 d;果肉可溶性固形物含量22.3%,总糖含量19.6 mg/100 g,维生素C（Vc）含量16.38 mg/100 g,可滴定酸含量0.43%;果实具有较好的耐贮性,催熟后在室温下可保存3～5 d。2012～2015年在海南、广东及广西进行种植试验,桂蕉9号对由FOC4引起的枯萎病表现出抗（耐）性,1代及2代香蕉植株的田间发病率为0.95%～52.30%,比对照品种（巴西蕉、桂蕉6号、桂蕉1号）减少7.38%～88.70%（绝对值）;1代及2代蕉单株平均产量为19.8～29.6 kg,总产量17001.0～74485.4 kg/ha,比对照品种增产-2.1%～515.5%。桂蕉9号于2015年6月通过广西农作物品种审定委员会审定。【结论】桂蕉9号是广西首个自主育成的抗（耐）香蕉枯萎病新品种,适宜在广西、云南、海南等香蕉主产区种植。

镰刀菌酸毒素对西瓜幼苗根细胞跨膜电位及叶细胞有关抗逆酶的抑制

【目的】了解引起连作西瓜严重枯萎病的尖孢镰刀菌毒素对西瓜幼苗根和叶细胞有关生理功能的影响。【方法】用从尖孢镰刀菌西瓜专化型菌株提取的真菌毒素镰刀菌酸处理西瓜幼苗,考察其对细胞根系跨膜电位和叶片中相关抗逆酶的影响。【结果】高浓度镰刀菌酸处理西瓜幼苗12h后,根部细胞跨膜电位比对照下降61.9%～81.8%。不同浓度的毒素处理24h后,根系脱氢酶活性减少35.9%～90.9%。镰刀菌酸明显加速了叶片细胞膜脂质过氧化,24h后叶片丙二醛含量是对照的5.2～11.0倍。叶片中的苯丙氨酸解氨酶活性先增加,然后减少,6～12h活性最高,为对照的7.2～10.5倍。过氧化氢酶、超氧化物岐化酶、过氧化物酶也是先上升然后下降。过氧化氢酶活性在毒素处理3h后达到高峰,但再过3h后下降18.6%～52.5%。超氧化物岐化酶和过氧化物酶在毒素处理12h后达到最大,然后下降。叶片中的β-1,3-葡聚糖酶和几丁质酶在毒素处理后先快速上升,然后下降,最大活性分别出现在处理后12h和3h,分别是对照的10和15～30倍。【结论】镰刀菌酸强烈抑制了西瓜幼苗的根和叶细胞正常生理功能。破坏了叶片细胞的正常防卫系统和根细胞吸水和运输功能,引起细胞膜脂质过氧化生成大量的丙二醛,细胞结构遭受严重破坏,导致发病甚至细胞死亡。

西瓜枯萎病菌专化型鉴定及防治研究

1982～1987年间自黑龙江、北京等10个省市采集到60份西瓜病株,分别进行分离和培养鉴定,结果有尖孢镰刀菌、茄病镰刀菌、木贼镰刀菌。致病性试验是将3个镰刀菌分别接种于西瓜品种郑州3号,结果尖孢镰刀菌致病,茄病镰刀菌和木贼镰刀菌不侵染。分别从黄瓜和甜瓜枯萎病株分离出来的尖孢镰刀菌,与西瓜病株分离得的尖孢镰刀菌进行交互接种于西瓜、甜瓜和黄瓜的幼苗上。鉴定的结果是不同的专化型,即尖孢镰刀菌西瓜专化型〔Fusarium oxysporum. f. sp. niveum(E. F. Smith)Snyder et. Hans.〕。在防治上,采用综合防治措施,经过1989～1990年试验,防效达90.91％～92.70％,增产58.95％～64.93％。

西瓜枯萎病菌遗传分化研究

本试验利用21个RAPD随机引物对50个西瓜枯萎病菌系进行了RAPD扩增，研究了病菌遗传多样性。结果表明，对供试菌系扩增出134条带，其中多态性带113条，占总带数的84．3％。菌系间的遗传相似系数变化在0．45-0．93之间，多数菌系间的同源程度较低。基于RAPD标记聚类分析表明，50个菌系划分为6个RAPD群（RAPD groups，简称RGs），即RGⅠ，RGⅡ，RGⅢ，RGⅣ，RGV和RGⅥ。其中RGⅠ和RGⅡ各含有一个菌系，分别来自秦皇岛和新疆；RCⅢ和RGⅤ包括2个菌系，分别来自秦皇岛、承德和廊坊、沧州；RGⅣ包括唐山、石家庄、邯郸和新疆菌系；其余的菌系属于RGⅥ。分类结果进一步说明西瓜枯萎病菌间存在着较大的遗传分化。

硝/铵营养对香蕉生长及其枯萎病发生的影响

通过水培试验,研究了不同硝/铵配比对香蕉生长及其根际尖孢镰刀菌侵染的影响。结果表明,1)铵硝混合营养对香蕉生长的效果优于单一营养,尤其是在75%硝态氮+25%铵态氮处理下香蕉生长最好,叶片中氮、磷、钾含量也最高;2)香蕉根际pH在100%铵态氮处理时最低,随着硝态氮比例的增加,pH逐渐上升;3)接种尖孢镰刀菌后,根际病原菌数量在100%铵态氮处理时最多,但是在根系细胞内却没有检测到,相反,随着硝态氮比例的增加,虽然在根际中检测到的病菌数量有所降低,但是在根系内均发现存在病原菌。本研究结果说明,相对于铵硝混合营养,全铵营养会导致香蕉生长受到一定的影响,但是却能够防止香蕉尖孢镰刀菌侵染进植物根系。由于在离体培养时,全铵可以抑制尖孢镰刀菌穿透植物细胞壁的过程,因此全铵培养植物时,其根部质外体及细胞中铵态氮浓度高很可能是抑制病原菌侵染的主要原因。

香蕉枯萎病的离体叶片接种技术研究

香蕉枯萎病是一种毁灭性真菌病害,对香蕉的种植和产业发展危害严重,其病原菌为尖孢镰刀菌古巴专化型(Fusariumoxysporum f.sp.cubense)。为研究该病原菌的致病机理和香蕉的枯萎病抗性机理,对香蕉枯萎病的离体叶片接种方法进行了研究,结果表明:香蕉叶片在离体条件下必须要有伤口才能被侵染发病,枯萎病菌4号小种的菌丝和分生孢子溶于无菌水或者PDB溶液都能引起发病;在一定浓度范围内,发病程度与刺孔数量和细菌浓度成正比;在刺孔条件下,香蕉枯萎病分生孢子浓度为0.9×105个/mL时的接种效果比较显著。

香蕉枯萎病的发生及防控研究现状

香蕉是世界第 2 大水果作物,也是世界贸易量最大的水果。由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp.cubense,Foc)引起的香蕉枯萎病是香蕉生产上最重要的病害之一,极大地限制了世界香蕉产业的健康和可持续发展。近年来国内外学者在其病害发生、病原生物学、侵染过程、病害流行、基因组测序、香蕉与 Foc 互作以及病害防控等方面开展了大量研究,但鲜见较为系统和全面的评述。本文就香蕉枯萎病的发生历史和危害现状、病原小种和遗传多样性以及防控方法等进行较为全面的梳理和总结,以期为该病害的研究和防控提供一定的参考。

农杆菌介导的香蕉枯萎病菌4号生理小种转化体系的优化

香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一,严重威胁和影响着香蕉产业的发展。本文针对香蕉枯萎病病原菌的4号生理小种,建立了农杆菌介导的转化体系,确定了影响转化效率主要因子的优化体系是:农杆菌在IM培养基诱导前农杆菌OD600为0.15、农杆菌经IM液体培养基诱导的时间为7h、乙酰丁香酮(AS)浓度为150μmol/L、Focr4孢子浓度为1×106个/mL、共培养时间为48h、培养温度为25℃、诱导培养基pH值为5.5。在此条件下,转化效率能达到700～800个转化子/106个香蕉枯萎病菌孢子。PCR验证表明外源的T-DNA已经成功随机地整合到该病原菌基因组中。目前,应用该转化体系已获得2300多个转化子,为后续克隆相关致病基因打下了良好基础。

香石竹枯萎病菌的生物学特性研究

对尖孢镰刀菌石竹专化型(Fusarium oxysporumf.sp.dianthi)的生物学特性进行了研究。结果表明,菌落在所供试的培养基上均能够生长,能有效地利用各种碳氮源,以蛋白胨为氮源和可溶性淀粉为最适合碳源。菌落在4-35℃范围均能生长,最适合温度为25℃。在pH 2-9的范围内都能生长,最适合pH 6。全光照条件下生长得最好,全黑暗的条件下生长得最慢。孢子的致死温度为65℃,10 min。生物学特性显示,该菌是一类对营养需求不高,对环境适应力强的病原菌。

枯草芽孢杆菌TG26防病增产效应的研究

枯草芽孢杆菌TG26分离自丝瓜根部土壤,对多种植物病原菌有强烈抑菌活性。在小麦感病品种盆栽试验中,对小麦赤霉病的防效为82.25～89.80％;浸根处理对西瓜枯萎病和烟草青枯病的苗期防效可达100％。田间小区试验结果,该菌剂对小麦赤霉病、西瓜枯萎病和烟草青枯病的防效分别为76.4％、73.1％和79.6％,并有明显的增产效应。其分泌的抗菌蛋白对植物病原菌有很强的抑菌作用。

甲基营养型芽孢杆菌对西瓜枯萎病菌拮抗作用的研究

在西瓜枯萎病重病园区,从生长正常西瓜根际分离获得一株细菌XG-1。经对峙培养和孢子萌发抑制测定,XG-1对西瓜枯萎病菌菌丝生长、孢子萌发具有良好抑制效果。XG-1明显抑制西瓜枯萎病菌菌丝向四周均匀扩展,抑菌带宽度平均为0.6 cm。XG-1培养液处理病原菌菌丝体和分生孢子培养液后,可使尖孢镰刀菌小型分生孢子由椭圆形致畸膨大数倍后成球形,使病原菌大型分生孢子由镰刀形畸变成葫芦串形,使菌丝体致畸膨大成串珠状,同时使分生孢子在合适的条件下不能萌发或萌发后生长缓慢,产生很短的芽管,而失去侵染力。田间小区试验表明,生防菌灌根处理西瓜苗死亡率为10%,相比对照处理的24.44%,能显著降低植株发病率。结合形态学、生理生化特征和16S r DNA序列分析将XG-1鉴定菌株为甲基营养型芽孢杆菌(Bacillus methylotrophicus)。

成都平原不同地区西瓜枯萎病菌致病力差异研究初探

对从成都平原的双流、青白江和德阳旌阳区3个不同西瓜主产区采集到的西瓜枯萎病菌进行病菌分离培养,得到3株西瓜枯萎病菌菌株。以中等抗性的西瓜品种"京欣"为寄主进行致病力测定,结果表明不同菌株的致病力差异达到了极显著水平,致病力最强的是来自德阳的Fon3,其次是青白江Fon1和双流Fon2。