辣椒疫病生防菌Penicillium striatisporum Pst10的筛选、抗菌物质分离纯化及分子标记
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摘要
辣椒疫病是由辣椒疫霉菌(Phytophthora capsici)引起的、世界范围内普遍发生的、毁灭性土传病害,20世纪90年代曾在中国20多个省连续爆发,造成了巨大的经济损失。由于疫霉菌特殊的生物学性状及其所在的土壤生态环境的复杂性,该病害的化学防治一直较为困难。尽管有许多用生防微生物控制该病害发生的报道,但防效均不理想。
本研究从挖掘生防微生物资源、获得天然抗菌化合物角度出发,开展了从各种生境中进行辣椒疫霉拮抗菌的筛选、盆栽防效试验、拮抗物质生成的最适培养条件确定、拮抗物质的分离纯化、鉴定及分子标记研究,取得如下结果。
以辣椒疫霉为靶标病原菌,从连作辣椒不同年限的土壤、用不同原料及制作工艺制得的堆肥、各种蔬菜作物体内等各种生态环境中筛选到辣椒疫霉的拮抗菌205株,其中拮抗真菌32株,拮抗放线菌78株,拮抗细菌95株,其中内生芽孢细菌37株。将在室内平板上拮抗作用强、持续时间长的放线菌A576、A329、芽孢细菌B105、BS211、真菌Tpc、Pst10进行液体培养,通过各菌株无菌培养滤液(sterile liquid culture filter,SLCF)对辣椒疫霉的抑菌活性及各菌株菌体及液体培养物对辣椒疫病的盆栽防效比较,显示真菌Pst10有较好的辣椒疫病生防潜力。经中国农业科学院微生物菌种保藏中心鉴定,Pst10为青霉稀有种Penicillium striatisporum。首次报道在中国境内分离到该种青霉,并将其应用于植物病害的生物防治。
抗菌谱测定表明青霉Pst10对P. capsici、P. infestans、P. drechsleri、P. nicotianae、 P. megasperma五种疫霉菌的菌丝生长均有强烈的抑制作用,拮抗带宽度30mm,并且对Cladosporium cucumerium和Sclerotinia sclerotiorum也有较强的拮抗作用,且在室内平板上,拮抗带30天不消失。Pst10液体培养制备的SLCF能杀死辣椒疫霉菌丝细胞,抑制孢子囊的形成,抑制孢子囊及孢子的萌发,SLCF稀释10-100倍仍能使辣椒疫霉菌丝呈现直径明显增粗、菌丝前端扭曲、菌丝分枝增加、分枝菌丝节间缩短等畸形症状。在辣椒基质盆栽实验中,单独施用Pst10的SLCF,在6天内能完全抑制疫病的发生,13天后,防效仍保持45%。在菜园土盆栽实验中,有机肥能明显促进Pst10在辣椒根际的定殖：盆钵栽苗10d后,青霉Pst10+有机肥处理中,辣椒根际青霉Pst10的数量是单独施加青霉Pst10处理的3倍；20d后上升为17倍。接种后40d,青霉Pst10在根际的数量仍有2.55×104cfu/g干土,占根际真菌总数的53%,有很强的定殖及保持优势种群的能力。青霉Pst10不同处理的防病实验结果显示：SLCF+Pst10+有机肥的处理对辣椒疫病的防效最好,移栽后14天,疫病发病率也仅有10%,对照发病率为100%。
Pst10在5种固体培养基上的菌丝生长速度、分泌物及孢子产生数量等生物学形状有很大差别,OAT是最适宜的产孢培养基。Pst10在9种液体培养基中的生物量、菌丝球形状及SLCF的抗菌活性也有显著性差异。综合考虑菌丝生物量和SLCF的抗菌活性,PEDB是Pst10最适宜的液体培养基。在9种培养基中都能生长,在Vogel及Jackson培养基中生长良好但却没有抗菌物质生成,可见菌丝生长和抗菌物质生成之间没有必然相关性。抗菌物质生成的培养条件研究表明,接种量对抗菌物质生成没有影响,Pst10在20-37℃温度范围内都能代谢产生拮抗物质,但28℃是最适合的温度。装液量对抗菌物质的生成有影响,250mL三角瓶中装20-100mL液体培养基对青霉Pst10代谢产生抗菌物质没有明显差异,装液超过100mL,抗菌物质的生成减少,说明Pst10产生抗菌物质需要氧气。在最适培养条件下,Pstl0从第4天开始大量产生抗菌物质,第8天达到高峰,高峰期维持7-8天。
对SLCF的抗菌活性检测方法开展了研究。在SLCF制备中发现,当接种量为1-4个菌饼时,用细菌滤膜过滤制备SLCF的抗菌活性显著低于用高速离心法获得SLCF的活性,部分抗菌物质被吸附在滤膜上,但这种差异随着接种量的增加而减小。推测可能接种量对Pst10的SLCF(?)勺总体活性没有明显影响,但对Pst10产生的抗菌物质的种类有影响：接种量小时,Pst10产生的抗菌物质中,易被细菌滤膜吸附的抗菌物质占较大比例；接种量大时,Pst10产生较多不被滤膜吸附的抗菌物质。由此可见Pst10代谢产生的抗菌物质是复杂多样的。采用平板打孔-抑菌圈法、含毒平板-菌丝生长抑制法及SLCF直接滴加三种方法对SLCF的抗菌活性进行检测,研究发现SLCF直接滴加法最灵敏；研究还发现,活性检测所用的培养基种类对检测结果有明显影响,SLCF在PDA上的抗菌活性大于在V8培养基上的活性。
与平板法检测抗菌物质相比,薄层层析法(TLC)检测能获取更多信息,因为在检测的同时就对抗菌物质进行了初步的分离。通过观察薄层层析板上抑菌圈的数量及位置就可初步判断抗菌物质的大概有几大类及各类抗菌物质的相对极性强弱。
将Pst10的PEDB培养液经纱布过滤,把滤液离心、真空浓缩100倍后用乙酸乙酯萃取获得抗菌物质粗提物；该粗提物经TLC分离后,在254nm紫外光线下观察到6条带,分别将6条带中的化合物回收后用辣椒疫霉进行生物测定,结果显示只有条带5有抗菌活性；将从条带5中回收的抗菌物质经硅胶柱层析,洗脱液中第5-8管有抗菌活性,将4管洗脱液合并浓缩后经HPLC分离,初步共收集到4组有活性的组分,改变洗脱条件,凸显有活性的洗脱峰,用HPLC制备型分离柱收集获得2个含量最大的活性组分fraction1和fraction5；将组分1和组分5进行'HNMR和13CNMR及二维NMR分析、紫外吸收光谱扫描,最后鉴定为Calbistrin A和CalbistrinB,二者互为同分异构体,分子式C31H40O8,分子量540.26。在中间每个分离步骤所得的活性物质中都能检测到Calbistrin A和Calbistrin B存在,说明二者均是由Pst10代谢产生。
对常见微生物的生物活性测定显示：Calbistrin A和Calbistrin B对假单胞菌细菌没有活性,但对芽孢细菌有抗生活性,且Calbistrin B的活性显著高于Calbistrin A. Calbistrin A和Calbistrin B对辣椒疫霉孢子萌发的MIC分别为7.5μg/mL和1.25μg/mLCalbistrin B的活性是Calbistrin A的6倍。种子发芽实验显示,Calbistrin B的浓度对萝卜种子的发芽率及根长没有显著影响。
采用根癌农杆菌介导的方法获得Pst10的潮霉素B抗性标记、遗传稳定的转化子41株,转化率为0.82%。所有转化子的生长速度和抗菌性能与野生型没有明显差异；获得12个绿色荧光蛋白-遗传霉素抗性(GFP-Gen)(?)双标记菌株,转化率0.24%,获得14个黄色荧光蛋白-遗传霉素(YFP-Gen)抗性双标记菌株,转化率0.28%。筛选到生长速度和拮抗性能与野生型没有明显差异的双标记转化子,为该菌株的专利保护实现了分子标记。
Phytophthora root rot (PRR), caused by Phytophthora capsici Leonian,is a common and destructive disease of greenhouse and field-grown chilli peppers(Capsicum frutescens L.) in China and the worldwide. The disease caused serious economic losses of chilli peppers in more than20Provinces of China. It has been difficult to control PRR with chemical methods since the special biologial characteristics of pathogen of Phtophthora sp. and complicated condition of soil. The biocontrol efficacy of disease suppression of PRR could not meet the requirement of agricultural production although there are a few reports about application of various of biocontrol agents that were used for disease control.
In order to exploit new microorganism for disease control of crops and obtain natural and microbial-soured chemicals against plant pathogens, we carried out some research works as following:
Two hundred and five antagonistic microbes against P.capsici were selected from soil which grew chilli pepper continuously in past decades, composting which was made with different raw material and composting technique and plant of varieties of vegetables crops, among them there were32antagonistic fungi,78antagonistic actinomyces and95antagonistic bacteria. Strain A576,A329,B105,BS211,Tpc and-Pst10showed strongly and lasting antagonism against P.capsici on plates in vivtro and was cultured in liquid medium respectively.The antagonistic activity of SLCF of each culture was examined and the effects on disease controlof PRR in pot experiment were compared. The results showed PstlO was better than other microbes and could be a potential candidate for PRR control. PstlO was identified as Pencillium striatisporum, a rare species, by Agricultural Culture Collection of China (ACCC) and named as PstlO in this research.This is the first study that reported Penicillium striatisporum was isolated in China and applied as biocontrol agent for disease control of plants.
PstlO showed very high antagonistic effects on mycelium growth of Phytophthora spp., Cladosporium cucumerium, and Sclerotinia sclerotiorum, and the inhibition zone was30mm and lasted more than30days in in vitro assays. The toxicity of sterilized liquid culture filtrates (SLCF) of Pst10grown in potato dextrose broth (PDB) was tested against Phytophthora capsici mycelium growth and sporangia/spore formation or germination. The SLCF completely inhibited mycelium growth and even at a100-fold dilution led to abnormal mycelium. The disease incidence of PRR was zero after6days transplantation in pot experiment with substrate treated with SLCF and still45%plants were healthy after13days transplantation. Composted pig manure slightly increased the colonization of Pst10in the chilli rhizosphere. In pot tests, the number of Pst10in chilli rhizosphere with treatment of Pst10and organic fertilizer was three folds of that in treatment of Pst10singly after10days of transplantation and the number increased to17folds after20days of transplantation. Strain PstlO showed strong ability of colonization and maintainment of dominatnt group since the number of Pst10in chilli pepper rhizosphere remained2.55×104cfu/g after40days. The lowest incidence of PRR was in the treatment consisting of SLCF, conidial suspension of Pst10and organic fertilizer with10%plants were infected by P.capsici after14days of transplantation, while the disease incidence of control was100%.
There were obvious difference about growth and secretes of mycelium and conidia production of Pst10when cultured on5different solid medium respectively, and OAT was optimum medium for conidia production. Also, the diffence of biomass of mycelium, sphere appearance of mycelium and antibiotic activity of SLCF was significant when Pst10was cultired in9kinds of different liquid media. PEDB was optimum medium when considering biomass of mycelium and bioactivity of SLCF. Pst10grew well in9types of media. The SLCF of Pst10cultured in Vogel or Jackson medium had no antagonistic activity against P.capsici although Pst10displayed favourable growth in this two media. There was no corelation between mycelium growth and antibiotics production. The studies on culture conditon for antibiotics production showed that:inoculum had no impact on antibiotics prodcution, The antibiotics could be produced by Pst10at20-37℃,and28℃was the best temperature.Gas flux could influence production of antibiotics of Pst10,butthe difference was not significant when the flask of250mL was filled with20-100mL medium.The antibiotics was produced in a large amount by Pst10after4days incubation and the antagonistic activity was highest on the8th days of incubation and this activity lasted for7-8days.
The methods for activity examination of SLCF were studied. The antagonistic activity of SLCF prepared by centrifugation with high speed was higher than that prepared by filtration through0.22μm Millipore membrane when the inoculum of Pst10was1-4disks in100mL medium contained in250mL flask, and the following experiment showed part of chemicals were adhered to Millipore membrane But the activity disparity between the two methods reduced with inoculum increasement. This result maybe suggest that the total activity of SLCF was not impacted by inoculum, while the varities of chemicals produced by Pst10were influenced by inoculum,that means Pst10produced much more antagonistic chemicals which were adhered to Millipore membrane with small inoculum,and more chemicals which were easily passed through Millipore membrane were produced by Pst10when with a large amount of inoculum. So, varieties of antifungal chemicals were produed by Pst10. The method of direct dropping of SLCF was the most sensitive among three methods of plate-hole-inhibiton zone, plate containing antibiotics-growth inhibition of mycelium and dropping SLCF directly. We also find antibiotic activity was affected by medium composition.
Compared with plate method used for activity examination, the TLC method suggested more information about antibiotics since chemicals could be seperated primarily at the same time of activity examination. We could know how many types of chemicals and the polarity of antibiotic chemicals exsisted in SLCF by numbers, size and position of inhibition zone on TLC plate.
Liquid cultures of Pst10grown in PEDB were filtered through two-layers of cheesecloth in a funnel and centrifuged,and the supernatants were collected and concentrated at45℃on a rotary evaporator to a final volume with1%of SLCF. The concentrated supernatants were extracted with ethyl acetate and consequently the antagonistic fraction was seperated by TLC. Under254UV,6bands were observed and the chemicals contained in each band were recovered.The bioassay results of chemicals recovered from each band showed the5th band contained antagonistic chemicals. The chemicals coming from the5th band was seperated by Silica column and elute was collected in tube. The elutes in the5-8th tubes had antagonism against P.capsici. The elutes in the four tubes was combined and concentrated and then fractioned by HPLC. Four fractions with antibiotic activity were obtained. Two purified fractions, named fractionl and fraction5, were collected in a large amount when HPLC condition was changed. The two fractions were analysized by1HNMR,13CNMR, dimension NMR, UV spectrum and finally were identifed as Calbistrin A and Calbistrin B respectively,which were isomer with formula of C31H40O8and molecular weight of540.26.
Calbistrin A and Calbistrin B had activity against Bacillus sp.,but had no activity against Pseudomonas sp. and Actinomyces sp.tested. The activity of Calbistrin B is higher than that of Calbistrin A. The MIC of Calbistrin A and Calbistrin B was7.5μg/mL and1.25μg/mL seperately. Calbistrin B had no toxicity to seed germination and root growth when the concentration applied was lower than10μg/mL which was8folds of MIC to P.capsici.
Forty-one transformants with Hygromycin B resistance marker were obtained with Agrobacterium tumefaciens-mediated transformation (ATMT), and the transformation rate was0.82%. There was no difference between wild type and all the transformants of Pst10about growth and antagonism against P. capsici. Twelve transformants with GFP-Geneticin resistance dual marker were obtained with ATMT with0.24%of transformation rate. Fourteen transformants with YFP-Geneticin resistance dual marker were obtained with ATMT with0.28%of transformation rate. There was no obvious difference about growth and antagonism against P. capsici between wild types and several transformants with GFP/YFP-Gen dual markers and the green or yellow fluorescent protein was expressed normally, which resulted in a molecular marker for patent protection of Pst10.
本研究从挖掘生防微生物资源、获得天然抗菌化合物角度出发,开展了从各种生境中进行辣椒疫霉拮抗菌的筛选、盆栽防效试验、拮抗物质生成的最适培养条件确定、拮抗物质的分离纯化、鉴定及分子标记研究,取得如下结果。
以辣椒疫霉为靶标病原菌,从连作辣椒不同年限的土壤、用不同原料及制作工艺制得的堆肥、各种蔬菜作物体内等各种生态环境中筛选到辣椒疫霉的拮抗菌205株,其中拮抗真菌32株,拮抗放线菌78株,拮抗细菌95株,其中内生芽孢细菌37株。将在室内平板上拮抗作用强、持续时间长的放线菌A576、A329、芽孢细菌B105、BS211、真菌Tpc、Pst10进行液体培养,通过各菌株无菌培养滤液(sterile liquid culture filter,SLCF)对辣椒疫霉的抑菌活性及各菌株菌体及液体培养物对辣椒疫病的盆栽防效比较,显示真菌Pst10有较好的辣椒疫病生防潜力。经中国农业科学院微生物菌种保藏中心鉴定,Pst10为青霉稀有种Penicillium striatisporum。首次报道在中国境内分离到该种青霉,并将其应用于植物病害的生物防治。
抗菌谱测定表明青霉Pst10对P. capsici、P. infestans、P. drechsleri、P. nicotianae、 P. megasperma五种疫霉菌的菌丝生长均有强烈的抑制作用,拮抗带宽度30mm,并且对Cladosporium cucumerium和Sclerotinia sclerotiorum也有较强的拮抗作用,且在室内平板上,拮抗带30天不消失。Pst10液体培养制备的SLCF能杀死辣椒疫霉菌丝细胞,抑制孢子囊的形成,抑制孢子囊及孢子的萌发,SLCF稀释10-100倍仍能使辣椒疫霉菌丝呈现直径明显增粗、菌丝前端扭曲、菌丝分枝增加、分枝菌丝节间缩短等畸形症状。在辣椒基质盆栽实验中,单独施用Pst10的SLCF,在6天内能完全抑制疫病的发生,13天后,防效仍保持45%。在菜园土盆栽实验中,有机肥能明显促进Pst10在辣椒根际的定殖：盆钵栽苗10d后,青霉Pst10+有机肥处理中,辣椒根际青霉Pst10的数量是单独施加青霉Pst10处理的3倍；20d后上升为17倍。接种后40d,青霉Pst10在根际的数量仍有2.55×104cfu/g干土,占根际真菌总数的53%,有很强的定殖及保持优势种群的能力。青霉Pst10不同处理的防病实验结果显示：SLCF+Pst10+有机肥的处理对辣椒疫病的防效最好,移栽后14天,疫病发病率也仅有10%,对照发病率为100%。
Pst10在5种固体培养基上的菌丝生长速度、分泌物及孢子产生数量等生物学形状有很大差别,OAT是最适宜的产孢培养基。Pst10在9种液体培养基中的生物量、菌丝球形状及SLCF的抗菌活性也有显著性差异。综合考虑菌丝生物量和SLCF的抗菌活性,PEDB是Pst10最适宜的液体培养基。在9种培养基中都能生长,在Vogel及Jackson培养基中生长良好但却没有抗菌物质生成,可见菌丝生长和抗菌物质生成之间没有必然相关性。抗菌物质生成的培养条件研究表明,接种量对抗菌物质生成没有影响,Pst10在20-37℃温度范围内都能代谢产生拮抗物质,但28℃是最适合的温度。装液量对抗菌物质的生成有影响,250mL三角瓶中装20-100mL液体培养基对青霉Pst10代谢产生抗菌物质没有明显差异,装液超过100mL,抗菌物质的生成减少,说明Pst10产生抗菌物质需要氧气。在最适培养条件下,Pstl0从第4天开始大量产生抗菌物质,第8天达到高峰,高峰期维持7-8天。
对SLCF的抗菌活性检测方法开展了研究。在SLCF制备中发现,当接种量为1-4个菌饼时,用细菌滤膜过滤制备SLCF的抗菌活性显著低于用高速离心法获得SLCF的活性,部分抗菌物质被吸附在滤膜上,但这种差异随着接种量的增加而减小。推测可能接种量对Pst10的SLCF(?)勺总体活性没有明显影响,但对Pst10产生的抗菌物质的种类有影响：接种量小时,Pst10产生的抗菌物质中,易被细菌滤膜吸附的抗菌物质占较大比例；接种量大时,Pst10产生较多不被滤膜吸附的抗菌物质。由此可见Pst10代谢产生的抗菌物质是复杂多样的。采用平板打孔-抑菌圈法、含毒平板-菌丝生长抑制法及SLCF直接滴加三种方法对SLCF的抗菌活性进行检测,研究发现SLCF直接滴加法最灵敏；研究还发现,活性检测所用的培养基种类对检测结果有明显影响,SLCF在PDA上的抗菌活性大于在V8培养基上的活性。
与平板法检测抗菌物质相比,薄层层析法(TLC)检测能获取更多信息,因为在检测的同时就对抗菌物质进行了初步的分离。通过观察薄层层析板上抑菌圈的数量及位置就可初步判断抗菌物质的大概有几大类及各类抗菌物质的相对极性强弱。
将Pst10的PEDB培养液经纱布过滤,把滤液离心、真空浓缩100倍后用乙酸乙酯萃取获得抗菌物质粗提物；该粗提物经TLC分离后,在254nm紫外光线下观察到6条带,分别将6条带中的化合物回收后用辣椒疫霉进行生物测定,结果显示只有条带5有抗菌活性；将从条带5中回收的抗菌物质经硅胶柱层析,洗脱液中第5-8管有抗菌活性,将4管洗脱液合并浓缩后经HPLC分离,初步共收集到4组有活性的组分,改变洗脱条件,凸显有活性的洗脱峰,用HPLC制备型分离柱收集获得2个含量最大的活性组分fraction1和fraction5；将组分1和组分5进行'HNMR和13CNMR及二维NMR分析、紫外吸收光谱扫描,最后鉴定为Calbistrin A和CalbistrinB,二者互为同分异构体,分子式C31H40O8,分子量540.26。在中间每个分离步骤所得的活性物质中都能检测到Calbistrin A和Calbistrin B存在,说明二者均是由Pst10代谢产生。
对常见微生物的生物活性测定显示：Calbistrin A和Calbistrin B对假单胞菌细菌没有活性,但对芽孢细菌有抗生活性,且Calbistrin B的活性显著高于Calbistrin A. Calbistrin A和Calbistrin B对辣椒疫霉孢子萌发的MIC分别为7.5μg/mL和1.25μg/mLCalbistrin B的活性是Calbistrin A的6倍。种子发芽实验显示,Calbistrin B的浓度对萝卜种子的发芽率及根长没有显著影响。
采用根癌农杆菌介导的方法获得Pst10的潮霉素B抗性标记、遗传稳定的转化子41株,转化率为0.82%。所有转化子的生长速度和抗菌性能与野生型没有明显差异；获得12个绿色荧光蛋白-遗传霉素抗性(GFP-Gen)(?)双标记菌株,转化率0.24%,获得14个黄色荧光蛋白-遗传霉素(YFP-Gen)抗性双标记菌株,转化率0.28%。筛选到生长速度和拮抗性能与野生型没有明显差异的双标记转化子,为该菌株的专利保护实现了分子标记。
Phytophthora root rot (PRR), caused by Phytophthora capsici Leonian,is a common and destructive disease of greenhouse and field-grown chilli peppers(Capsicum frutescens L.) in China and the worldwide. The disease caused serious economic losses of chilli peppers in more than20Provinces of China. It has been difficult to control PRR with chemical methods since the special biologial characteristics of pathogen of Phtophthora sp. and complicated condition of soil. The biocontrol efficacy of disease suppression of PRR could not meet the requirement of agricultural production although there are a few reports about application of various of biocontrol agents that were used for disease control.
In order to exploit new microorganism for disease control of crops and obtain natural and microbial-soured chemicals against plant pathogens, we carried out some research works as following:
Two hundred and five antagonistic microbes against P.capsici were selected from soil which grew chilli pepper continuously in past decades, composting which was made with different raw material and composting technique and plant of varieties of vegetables crops, among them there were32antagonistic fungi,78antagonistic actinomyces and95antagonistic bacteria. Strain A576,A329,B105,BS211,Tpc and-Pst10showed strongly and lasting antagonism against P.capsici on plates in vivtro and was cultured in liquid medium respectively.The antagonistic activity of SLCF of each culture was examined and the effects on disease controlof PRR in pot experiment were compared. The results showed PstlO was better than other microbes and could be a potential candidate for PRR control. PstlO was identified as Pencillium striatisporum, a rare species, by Agricultural Culture Collection of China (ACCC) and named as PstlO in this research.This is the first study that reported Penicillium striatisporum was isolated in China and applied as biocontrol agent for disease control of plants.
PstlO showed very high antagonistic effects on mycelium growth of Phytophthora spp., Cladosporium cucumerium, and Sclerotinia sclerotiorum, and the inhibition zone was30mm and lasted more than30days in in vitro assays. The toxicity of sterilized liquid culture filtrates (SLCF) of Pst10grown in potato dextrose broth (PDB) was tested against Phytophthora capsici mycelium growth and sporangia/spore formation or germination. The SLCF completely inhibited mycelium growth and even at a100-fold dilution led to abnormal mycelium. The disease incidence of PRR was zero after6days transplantation in pot experiment with substrate treated with SLCF and still45%plants were healthy after13days transplantation. Composted pig manure slightly increased the colonization of Pst10in the chilli rhizosphere. In pot tests, the number of Pst10in chilli rhizosphere with treatment of Pst10and organic fertilizer was three folds of that in treatment of Pst10singly after10days of transplantation and the number increased to17folds after20days of transplantation. Strain PstlO showed strong ability of colonization and maintainment of dominatnt group since the number of Pst10in chilli pepper rhizosphere remained2.55×104cfu/g after40days. The lowest incidence of PRR was in the treatment consisting of SLCF, conidial suspension of Pst10and organic fertilizer with10%plants were infected by P.capsici after14days of transplantation, while the disease incidence of control was100%.
There were obvious difference about growth and secretes of mycelium and conidia production of Pst10when cultured on5different solid medium respectively, and OAT was optimum medium for conidia production. Also, the diffence of biomass of mycelium, sphere appearance of mycelium and antibiotic activity of SLCF was significant when Pst10was cultired in9kinds of different liquid media. PEDB was optimum medium when considering biomass of mycelium and bioactivity of SLCF. Pst10grew well in9types of media. The SLCF of Pst10cultured in Vogel or Jackson medium had no antagonistic activity against P.capsici although Pst10displayed favourable growth in this two media. There was no corelation between mycelium growth and antibiotics production. The studies on culture conditon for antibiotics production showed that:inoculum had no impact on antibiotics prodcution, The antibiotics could be produced by Pst10at20-37℃,and28℃was the best temperature.Gas flux could influence production of antibiotics of Pst10,butthe difference was not significant when the flask of250mL was filled with20-100mL medium.The antibiotics was produced in a large amount by Pst10after4days incubation and the antagonistic activity was highest on the8th days of incubation and this activity lasted for7-8days.
The methods for activity examination of SLCF were studied. The antagonistic activity of SLCF prepared by centrifugation with high speed was higher than that prepared by filtration through0.22μm Millipore membrane when the inoculum of Pst10was1-4disks in100mL medium contained in250mL flask, and the following experiment showed part of chemicals were adhered to Millipore membrane But the activity disparity between the two methods reduced with inoculum increasement. This result maybe suggest that the total activity of SLCF was not impacted by inoculum, while the varities of chemicals produced by Pst10were influenced by inoculum,that means Pst10produced much more antagonistic chemicals which were adhered to Millipore membrane with small inoculum,and more chemicals which were easily passed through Millipore membrane were produced by Pst10when with a large amount of inoculum. So, varieties of antifungal chemicals were produed by Pst10. The method of direct dropping of SLCF was the most sensitive among three methods of plate-hole-inhibiton zone, plate containing antibiotics-growth inhibition of mycelium and dropping SLCF directly. We also find antibiotic activity was affected by medium composition.
Compared with plate method used for activity examination, the TLC method suggested more information about antibiotics since chemicals could be seperated primarily at the same time of activity examination. We could know how many types of chemicals and the polarity of antibiotic chemicals exsisted in SLCF by numbers, size and position of inhibition zone on TLC plate.
Liquid cultures of Pst10grown in PEDB were filtered through two-layers of cheesecloth in a funnel and centrifuged,and the supernatants were collected and concentrated at45℃on a rotary evaporator to a final volume with1%of SLCF. The concentrated supernatants were extracted with ethyl acetate and consequently the antagonistic fraction was seperated by TLC. Under254UV,6bands were observed and the chemicals contained in each band were recovered.The bioassay results of chemicals recovered from each band showed the5th band contained antagonistic chemicals. The chemicals coming from the5th band was seperated by Silica column and elute was collected in tube. The elutes in the5-8th tubes had antagonism against P.capsici. The elutes in the four tubes was combined and concentrated and then fractioned by HPLC. Four fractions with antibiotic activity were obtained. Two purified fractions, named fractionl and fraction5, were collected in a large amount when HPLC condition was changed. The two fractions were analysized by1HNMR,13CNMR, dimension NMR, UV spectrum and finally were identifed as Calbistrin A and Calbistrin B respectively,which were isomer with formula of C31H40O8and molecular weight of540.26.
Calbistrin A and Calbistrin B had activity against Bacillus sp.,but had no activity against Pseudomonas sp. and Actinomyces sp.tested. The activity of Calbistrin B is higher than that of Calbistrin A. The MIC of Calbistrin A and Calbistrin B was7.5μg/mL and1.25μg/mL seperately. Calbistrin B had no toxicity to seed germination and root growth when the concentration applied was lower than10μg/mL which was8folds of MIC to P.capsici.
Forty-one transformants with Hygromycin B resistance marker were obtained with Agrobacterium tumefaciens-mediated transformation (ATMT), and the transformation rate was0.82%. There was no difference between wild type and all the transformants of Pst10about growth and antagonism against P. capsici. Twelve transformants with GFP-Geneticin resistance dual marker were obtained with ATMT with0.24%of transformation rate. Fourteen transformants with YFP-Geneticin resistance dual marker were obtained with ATMT with0.28%of transformation rate. There was no obvious difference about growth and antagonism against P. capsici between wild types and several transformants with GFP/YFP-Gen dual markers and the green or yellow fluorescent protein was expressed normally, which resulted in a molecular marker for patent protection of Pst10.
引文
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