灰霉病是保护地蔬菜栽培中危害十分严重的病害，传统的化学防治造成了严重的农药残留和环境污染。应用微生物进行生物防治能够克服上述缺点，是一种安全、有效和环保的方法。本研究旨在从黄瓜植株内分离、筛选能够抑制灰霉病的拮抗细菌，并测定拮抗菌株在不同处理下对灰霉病的防治作用。主要结果如下：通过平板对峙培养筛选出8株对黄瓜灰霉病菌具有较强拮抗作用的细菌，再对这8株菌发酵滤液的抑菌活性进行测定，选出抑制效果最好的两个菌株B12和B13，抑制率分别为84.0%和81.8%。在离体叶片上，B12、B13发酵滤液对灰霉病菌的扩展均有较强的抑制作用，抑制率分别为83.2%和81.4%。两菌株活性产物可引起病原菌菌丝扭曲，菌丝膨大成串珠状分枝，顶端膨胀后细胞壁破裂，原生质外溢，产生溶菌作用。B12、B13发酵液对于灰霉病菌孢子的萌发具有强烈的抑制作用。在孢子萌发试验中，经B12、B13发酵液处理的孢子萌发率仅分别为5.5%和8.6%，滤液对孢子萌发的抑制效果与发酵液相近。将两菌的发酵滤液置于不同的温度和pH环境下处理，当温度在100℃以下、pH值在6～9时，滤液的抑菌效果表现出较强的稳定性。在温室试验中，两菌发酵液对灰霉病有较好的预防效果，B12、B13的保护防效分别为70.5% 和68.7%，治疗防效分别为51.2%和47.8%。

枯草芽孢杆菌（Bacillus subtilis）是土壤和植物微生态的优势种群，内生芽孢，抗逆能力强，繁殖速度快，营养要求简单，对农作物安全。作为植物根际有益微生物，通过分泌抗生物质和生长竞争，在防治植物病害方面发挥多种有益作用[1]。剂型以活体芽孢为主，田间施用可以较长时间的发挥抑制病菌作用[2～3]。枯草芽孢杆菌Xi-55是本课题组从水稻植株上分离、筛选出来的一株活性较强的生防菌株，研究发现对多种植物病原菌具有良好的防治作用[4]。芽孢杆菌的发酵培养是工业化大规模生产芽孢杆菌制剂的前提。本文对所筛选出的枯草芽孢杆菌的发酵条件进行优化，进行20L发酵罐的放大培养研究，以提高其发酵水平，为芽孢杆菌制剂的工业化生产提供参考依据。1.1.1 供试菌株 枯草芽孢杆菌Xi-55，四川省农业科学院植物保护研究所分离获得。1.1.2 培养基 斜面培养基（LB培养基）：酵母膏5g，蛋白胨10g，NaCl 10g，琼脂15g，蒸馏水1000ml，pH值7.0。种子培养基（BPY培养基）：牛肉膏5g，蛋白胨10g，NaCl 5g，酵母膏 5g，葡萄糖 5g，蒸馏水1000ml，pH值7.0。基础发酵培养基（KB培养基）：蛋白胨 20g，甘油 10ml，K2HPO4 1.5g，MgSO4·7H2O 1.5g，蒸馏水1000ml，pH值7.0。1.2.1 培养方法 将保存在斜面上的菌种用接种环以划线形式接入LB平板上，28℃恒温培养24h活化。将活化的菌株接入装有100ml种子培养基的250ml三角瓶中，在28℃，180rpm条件下，振荡培养36～48h，制备液体种。按1%的接种量接入发酵培养基（100ml/250ml三角瓶），摇床振荡培养，测定发酵菌数量。1.2.2 生长量的测定 采用平板菌落记数法。培养液用10倍梯度稀释法稀释6～7个梯度（101，102，103，……，107）后，选择3个稀释度较高的梯度稀释液，分别吸取50μl在LB平板上，然后用灭菌的L形玻棒将菌液涂匀，每梯度重复3次。28℃恒温培养24～36h后，调查平板上的菌落数，然后计算活菌数（cfu/ml）[5]。1.2.3 培养基的优化 采用正交表L9（34）[6]，四因素三水平安排试验。1.2.4 发酵条件的优化 采用优化培养基通过单因子试验，测定不同时间、温度、初始pH值、接种量和装液量对发酵菌数的影响。1.2.5 扩大培养 采用德国产Biostat C 20L全自动液体发酵罐，配制发酵培养基10L，添加消泡剂CXX-910 0.5‰。种子培养及接种量均同摇瓶试验。发酵技术参数设为：发酵温度28℃±0.5℃，通气量10L/min，搅拌转速180rpm，溶氧控制设定为以通气量为主、转速为辅，罐压0.05～0.06MPa，初始pH值为7.2。每隔4h取样，测定发酵菌数和观察芽孢形成情况，同时记录罐体内pH值变化。分别以培养时间为横坐标，菌数和pH值为纵坐标，绘制枯草芽孢杆菌在发酵罐中培养的生长曲线和pH值曲线。采用L9（34）正交设计方案，分四因素三水平对Xi-55发酵培养，测定高峰期菌数，正交试验因素水平见表1，正交设计及结果见表2，极差分析见表3。结果表明：4种营养成分对其菌数影响的顺序是A＞C＞D＞B，培养基成分优化组合为A3B2C1D3，即蛋白胨3%，甘油1.0%，K2HPO4 0.05%，MgSO4·7H2O 0.15%。试验因素Experimentalfactor水平（Level）（%）123蛋白胨（Peptone）123甘油（Glycerine）0.51.01.5K2HPO40.050.100.15MgSO4·7H2O0.050.100.15表1 培养基优化正交试验因素水平Table 1 Orthogonal experimental factor levels for medium optimization试验号Experimentalnumble水平（Level）（%）A[蛋白胨]PeptoneB[甘油]GlycerineC[K2HPO4]D[MgSO4·7H2O]菌数（Bacteriumamount）（109cfu/ml）111117.01212225.25313331.834212318.585223110.92623129.927313211.428321325.259332123.00表2 培养基优化L9（34）正交设计及结果Table 2 L9（34） orthogonal design and results for medium opitizationA[蛋白胨]PeptoneB[甘油]GlycerineC[K2HPO4]D[MgSO4·7H2O]K14.7012.3315.6113.64K213.1413.8114.068.86K319.8911.588.0615.22R15.192.237.556.36优化组合Optimization-groupA3B2C1D3因素顺序FactororderA＞C＞D＞B表3 培养基优化L9（34）的极差分析Table 3 Range analysis of L9（34） for medium opitization2.2.1 时间对Xi-55发酵细菌数量的影响 每隔12h从摇床上取样，测定发酵液中细菌数量，绘制Xi-55生长曲线。结果显示（图1），在发酵0～12h 之间，菌体生长繁殖缓慢；12～36h 为菌体生长加速期，也为菌体繁殖高峰时期，发酵液内细菌数量明显增加；36～48h为稳定期，菌体数量基本稳定；60h以后为孢子衰亡期，活菌由于自身产生的分解物质而使菌体分解，发酵液变透明。根据该试验结果，36～48h为Xi-55适宜发酵时间。2.2.2 温度对Xi-55发酵细菌数量的影响 分别置于24℃、26℃、28℃、30℃和32℃摇瓶培养，测定不同培养温度的生长量，结果表明（图2），28℃细菌数量最高，28～30℃细菌数基本稳定，低于28℃和超过30℃菌数明显下降。所以28～30℃为Xi-55适宜发酵温度。图1 时间对发酵菌数的影响Figure 1 Effects of time on the bacteria amount图2 温度对发酵菌数的影响Figure 2 Effects of temperature on the bacteria amount2.2.3 初始pH值对Xi-55发酵细菌数量的影响 设定pH值分别为5，6，7，8，9的培养基摇瓶培养，测定细菌生长量。结果显示（图3），当pH值为7时，细菌数量最高，当pH值为 7～8时，细菌数量基本稳定；pH值低于7和超过8，菌数明显减少；且 pH值为9或5时，菌体数量急剧减少或为零，据此推断pH值高于9或低于5时，Xi-55生长可能严重受抑制甚至不能生长，有待进一步研究。所以发酵培养基的适宜初始pH值为7～8。2.2.4 接种量对Xi-55发酵菌数的影响 分别采用0.5%、1%、2%、3%、4%等5个接种量梯度摇瓶培养，测定细菌生长量，结果显示（图4），接种量在0.5%～2%范围内，菌量数随接种量的增加而增加，接种量为2%时，菌量数最高；接种量超过2%，菌数明显减少。总体上来看，菌体生长量并非随着接种量的增加而增加，而是有一个限度。所以，2%为最佳接种量。图3 初始pH值对发酵菌数的影响Figure 3 Effects of initial pH value on bacteria amount图4 接种量对发酵菌数的影响Figure 4 Effects of inoculation amount on bacteria amount2.2.5 装液量对Xi-55发酵菌数的影响 采用装液量分别为50ml/500ml、100ml/500ml、150ml/500ml、200ml/500ml锥形瓶摇床振荡培养，测定细菌生长量，试验结果表明，通过摇瓶装液量的不同来调节通气量对Xi-55发酵菌数有较大影响。一定范围内，装液量越少，则通气量越高，氧气供应越充足，那么细菌数量就越高。50ml与100ml装液量差别不明显，但从总生长量考虑，100ml/500ml锥形瓶为最佳装液量（图5）。图5 装液量对发酵菌数的影响Figure 5 Effects of pack amount on bacteria amount在摇瓶优化发酵条件的基础上，进行了20L发酵罐的扩大培养。从生长曲线（图6）可以看出，发酵罐扩大培养的发酵周期与摇瓶发酵周期基本一致。由于发酵罐的搅拌和通氧条件均优于摇瓶发酵，因此，菌体浓度和芽孢同步形成率都明显优于摇瓶发酵。36h菌体数量达到最高量，50.61×109cfu/ml；然后进入稳定期，开始大量形成芽孢，44h形成芽孢90%以上。分析pH值曲线（图6）发现，初始pH值7.2，随着菌体生长，pH值缓慢下降；对数生长期菌体迅速繁殖，pH值也急剧下降，表明菌体大量利用养分产生了酸性物质；稳定期随着芽孢逐渐形成，pH值回升，44～48h时，芽孢数量达到最大；52h以后pH值上升至7.4左右，可以看到菌体碎片，细胞自溶，表明生长和代谢受到抑制。因此，发酵终止应在52h前，最佳放罐时间应在44～48h。图6 20L发酵罐中的生长与pH值曲线Figure 6 The growth and pH curve in 20L fermentation tank从以上试验可以看出，培养基的组成和时间、温度、培养基初始pH值、接种量、通气量、摇床转速等培养条件均对菌株的生长有很大的影响。通过单因子试验和正交试验方法，确定枯草芽孢杆菌Xi-55优化培养基为：蛋白胨3%，甘油1.0%，K2HPO4 0.05%，MgSO4·7H2O 0.15%；优化发酵条件为：时间36～48h，温度28～30℃，初始pH值7～8，接种量2%，装液量100ml/500ml锥形瓶，摇床转速180～200rpm。并进行了发酵罐扩大培养，36h达到生长高峰期，最适放罐时间44～48h；此时所获得的菌体数量约为50亿个/ml，为大规模工业化发酵培养提供了有益借鉴。在发酵过程中发现，发酵液的黏度较大，很容易产生泡沫，必须用消泡剂来消除。而本试验中采用的发酵专用含硅消泡剂CXX-910对pH值有轻微影响，故添加消泡剂不宜过多。操作时可预先在培养基里添加少量消泡剂，然后通过发酵罐上的补料装置在发酵过程中自行控制补充，这样可以在满足消泡的同时尽量降低消泡剂的添加量，减少消泡剂对pH值的影响，比一次性添加或单纯通过补料装置添加效果要好。

烟草青枯病是由青枯菌（Ralstonia solanacearum）引起的一种细菌性病害，是烟草的一大毁灭性病害。此病主要分布于世界热带、亚热带及温带等湿热地区。我国烟草青枯病菌在长江以南发生居多，据报道，该病在多发病区旱地烟田的发病率为30%～50%。对烟草青枯病的防治，迄今仍无十分有效的措施，通常采用化学农药方法防治，但由于青枯病是维管束类病害，因而限制了化学农药的使用；而且化学防治易产生抗药性，造成环境污染。近年来，由于生物防治具有无污染、无公害、长效性等特点，所以青枯病的生物防治逐渐引起国内外的高度重视[1～4]。目前已取得了良好的进展，如魏春妹等[5]研制出番茄青枯病和烟草青枯病的生防制剂“青枯散”，田间试验证明该制剂对番茄青枯病、烟草青枯病有70%和80%的防治效果。为了筛选拮抗能力、定殖竞争能力强和效果稳定的优势菌株，本实验室从烟草的根标、根围土壤分离筛选出了拮抗细菌，并进行了室内拮抗能力的测定和盆栽烟苗防病的系列研究，得到了一株拮抗能力较好的菌株SH7，具有较好的商业化前景。本研究进行了SH7摇瓶发酵试验，筛选适合该菌株的发酵培养基配方和发酵条件。枯草芽孢杆菌SH7菌株由本室筛选、保存。种子培养基：牛肉汁液体培养基（酵母粉0.5g、葡萄糖10g、蛋白胨5g、牛肉膏3g、pH值7.0，定容至1L）。待测培养基的配制[6]：①号培养基：淀粉0.15%、葡萄糖0.5%、尿素0.1%、磷酸氢二钾0.3%、磷酸二氢钾0.15%、硫酸镁0.05%、酵母膏0.02%、氯化铁0.01%、碳酸钙0.01%、豆粕1%；②号培养基：淀粉0.15%、葡萄糖0.5%、尿素0.1%、磷酸氢二钾0.3%、磷酸二氢钾0.15%、硫酸镁0.05%、酵母膏0.02%、氯化铁0.01%、碳酸钙0.01%、豆粕1%、30.8mg/L的硫酸锰溶液0.1%；③号培养基：牛肉膏0.3%、蛋白胨1%、葡萄糖1%、氯化钠0.5%；④号培养基：牛肉膏0.3%、蛋白胨1%、葡萄糖1%、氯化钠0.5%、淀粉0.3%、30.8mg/L的硫酸锰溶液0.1%。将4种不同培养基pH值均调为7.2～7.3，装液量为60ml/300ml。121℃灭菌20min，备用。1.2.1 种子准备 牛肉汁液体培养基，pH值为7.0，装瓶量为100ml/300ml三角瓶，121℃灭菌20min。将牛肉汁液体培养基接菌后37℃过夜摇菌。1.2.2 培养基配方筛选 上述4种培养基中接种种子菌液1.5ml，在31℃条件下，摇菌36h后取5ml发酵完毕的菌液，在12000r/min下离心5min，倒掉上清，冷冻干燥沉淀18h后，称重量。1.2.3 发酵培养条件优化 每次只改变测定的一个发酵条件，其他方法同1.2.2。2 结果从表1可以看出，3次重复中5ml培养液中菌体干重的平均值大小依次为④号（0.0094g）＞③号（0.0089g）＞①号（0.0059g）＞②号（0.0054g），因此④号培养基较适合该菌的发酵。2.2.1 pH值 将发酵培养基的pH值分别调为7.0、7.3、7.6、7.9、8.2，3次重复。在装瓶量为60ml/300ml，接种量为1.5ml，31℃，180/min条件下摇菌36h，取5ml发酵液，冷冻干燥后称重。结果为，pH值7.0为0.0081g、pH值7.3为0.0061g、pH值7.6为0.0079g、pH值7.9为0.0082g、pH值8.2为0.0092g。pH值8.2比较适合该菌的发酵（图1）。重量（g）①号②号③号④号10.00590.00510.00970.013120.00660.00530.00970.006230.00510.00380.00720.0090平均0.00590.00540.00890.0094表1 培养基配方筛选结果2.2.2 装瓶量 将发酵培养基的pH值分别调为8.2，接种量为1.5ml，在300ml的三角瓶中分别装入40ml、60ml、80ml、100ml、120ml，3次重复。在31℃、180r/min条件下摇菌36h，取5ml发酵液，冷冻干燥后称重。结果为，40ml的为0.0065g、60ml的为0.0071g、80ml的为0.0065g、100ml的为0.0053g、120ml的为0.0062g。300ml的三角瓶中装60ml培养基较适合该菌生长（图2）。图1 pH值筛选结果图2 装瓶量筛选结果2.2.3 接种量 将培养基的pH值调为8.2，装瓶量为60ml/300ml三角瓶，接种量分别设为2%、4%、6%、8%、10%，3次重复。在31℃、180r/min条件下摇菌36h，取5ml发酵液，冷冻干燥后称重。结果是，接种量2%的为0.0098g、4%的为0.0108g、6%的为0.0103g、8%的为0.0112g、10%的为0.0096g。较合适的接种量为8%（图3）。2.2.4 培养温度 将培养基的pH值调为8.2，装瓶量为60ml/300ml三角瓶，接种量为8%，将温度分别设为25℃、28℃、31℃、34℃、37℃。在180r/min条件下摇菌36h，取5ml发酵液，冷冻干燥后称重。其结果是，25℃为0.0087g、28℃为0.0107g、31℃为0.0112g、34℃为0.0099g、37℃为0.0106g。较合适的温度为31℃（图4）。图3 接种量筛选结果图4 温度筛选结果2.2.5 培养转速 将培养基的pH值调为8.2，装瓶量为60ml/300ml三角瓶，接种量为8%，温度为31℃。将转速分别设为120r/min、150r/min、180r/min、210r/min、240r/min，摇菌36h，取5ml发酵液，冷冻干燥后称重。其结果是，120r/min为0.0091g、150r/min为0.0109g、180r/min为0.0092g、210r/min为0.0107g、240r/min为0.0101g。150r/min比较适合该菌发酵，所以选定150r/min作为较适转速（图5）。2.2.6 发酵时间 将培养基的pH值调为8.2，装瓶量为60ml/300ml三角瓶、接种量为8%、温度为31℃、转速为150r/min，将摇菌时间分别设为12h、24h、36h、48h、60h，取5ml发酵液，冷冻干燥后称重。其结果是，12h为0.0038g、24h为0.0062g、36h为0.0063g、48h为0.0067g、60h为0.0063g。发酵时间36h和60h菌体重量相同，但48h菌体重量最大，发酵时间越长对于生产越不经济，所以选取48h为较适发酵时间（图6）。图5 转速结果筛选图6 发酵时间筛选结果为了确定最优的培养基配方，我们将摇瓶试验结果相对较好的④号培养基作为基础培养基（培养基中其他营养成分不变），从中选出4种主要成分，采用优化好的发酵条件，按正交设计表L9（34）设计了4个因素、3个水平的正交试验，以进一步优化培养基配比[7，8]。正交试验设计和结果见表2。由表中R值可看出，影响SH7菌量依次为：牛肉膏＞蛋白胨＞NaCl＞葡萄糖。正交试验结果表明最佳培养基组合为：0.9%牛肉膏+2%蛋白胨+2%葡萄糖+1.1%NaCl。试验号牛肉膏（%）蛋白胨（%）葡萄糖（%）NaCl（%）菌体含量（g/5ml）1号1（0.3%）1（1%）1（1%）1（0.5%）0.009112号12（1.5%）2（1.5%）2（0.8%）0.010563号13（2%）3（2%）3（1.1%）0.011784号2（0.6%）1230.011445号22310.012566号23120.011897号3（0.9%）1320.011228号32130.012679号33210.01233Ⅰj0.03145g0.03177g0.03367g0.03400gⅡj0.03589g0.03579g0.03433g0.03367gⅢj0.03622g0.03600g0.03556g0.03589gR0.00477g0.00423g0.00189g0.00222g表2 正交试验设计及结果本文通过对该菌培养基配方和培养条件的优化选择以及正交试验，初步确定最适宜SH7菌株生长的培养基配方及培养条件如下：0.9%牛肉膏+2%蛋白胨+2%葡萄糖+1.1%NaCl；发酵起始pH值为8.2、300ml的三角瓶装瓶量为60ml、接种量为8%、温度为31℃、转速为150r/min，发酵时间为48h。这为SH7菌株的发酵罐放大试验提供了理论依据。本试验采用渐进法优化发酵条件，每确定一项，就在下一目标筛选中应用，这样使得得到的试验结果更准确。同时也要考虑到实际生产中的成本问题，例如某些营养物质含量不能太高或发酵周期不能过长，否则成本就会升高。众所周知，烟草业在国民经济中占有重要地位，目前青枯病的化学防治和生防都不甚理想，一定程度上制约了我国的烟草生产和出口。本实验室所筛选出的芽孢杆菌SH7对烟草青枯病菌表现出了良好的拮抗性，温室药效试验达到了较高的水平，前景广阔。本试验筛选出了适合该菌的发酵配方并优化了发酵条件，为该菌的生产应用打下了良好的基础。

小麦是渑池县的一种主要粮食作物。在小麦腥黑穗病的发生上，渑池县20世纪50～60年代比较普遍，80年代以后大部分地区已基本绝迹。近年来，小麦腥黑穗病在部分地区开始暴发，而且发生面积逐年加大，有大面积漫延之势。在小麦腥黑穗病的发生上，开始大都为零星发生，病株率较低，加上大部分群众对该病害不具备识别能力，早期不易引起群众的足够重视。一般经过2～3年病菌的传播、扩散和病菌的积累后，随着发病面积不断扩大和田间病穗率的提高。小麦腥黑穗病病穗比正常小麦麦穗落黄时间晚3～5天。当正常小麦麦穗已变黄时，小麦腥黑穗病病穗略显暗绿色，颖壳和麦芒稍向外张开，露出部分病粒。病粒比好麦粒粗短，初为暗绿色，以后变为灰黑色或淡灰色，外面包着一层灰白色膜，里面充满鱼腥味的黑粉（病菌的厚垣抱子），所以俗称腥乌麦或臭黑疸。病株一般比健株矮小，分蘖增多。小麦腥黑穗病病原菌有两种，一种是Tilletia caries（DC.）Tul.（小麦网腥黑粉菌），另一种是Tilletia foetida（Wallr.）Liro（称小麦光腥黑粉菌）。有报道Tilletia ntraversa Kühn称小麦矮腥黑粉菌也能引起腥黑穗病发生。两种病原菌均属担子菌亚门真菌。小麦网腥黑粉菌孢子堆生在子房内，外包果皮，与种子同大，内部充满黑紫色粉状孢子，具腥味。孢子球形至近球形，浅灰褐色至深红褐色，大小14～20μm，具网状花纹，网眼宽2～4μm。小麦光腥黑粉菌孢子堆同上。孢子球形或椭圆形，有的长圆形至多角形，浅灰色至暗榄褐色，大小15～25μm，表面平滑，也具腥味。小麦矮腥黑粉菌成群的孢子为暗黄褐色，分散的孢子近球形，浅黄色至浅棕色，大小14～18μm，具网纹，网脊高2～3μm，网目直径3～4.5μm，有的可达9.5～10μm，外面包被厚1.5～5.5μm的透明胶质鞘。主要引致小麦矮腥黑穗病。小麦脱粒时，病粒破裂，病菌孢子飞散，粘附在种子表面，调运带有小麦腥黑穗病病菌的种子是造成小麦腥黑穗病远距离传播的主要途径。群众相互间的种子串换是造成小麦腥黑穗病在一定区域内大面积扩散的主要原因。混有病菌的麦糠、麦秸、淘麦水等沤粪或喂牲口，使粪肥中带有病菌，施入麦地，也可以传病；若多户群众共用一个麦场，在小麦脱粒或晾晒时也可以传病；在个别寒冷干燥的地区，落在土中的病菌孢子存活时间较长，也可传病。病菌以厚垣孢子附在种子外表或混入粪肥、土壤中越冬或越夏。小麦播种后，当种子发芽时，粘附在种子表面或粪肥、土壤中的病菌孢子发芽并侵入小麦幼芽，厚垣孢子也随即萌发，厚垣孢子先产生先菌丝，其顶端生6～8个线状担孢子，不同性别担孢子在先菌丝上呈“H”状结合，然后萌发为较细的双核侵染线。从芽鞘侵入麦苗并到达生长点，后病菌在麦株内以菌丝体形态随小麦而发育，到孕穗期，侵入子房，最后到达花部，破坏花器正常发育，抽穗时在麦粒内形成菌瘿即病原菌的厚垣孢子，即为病粒。小麦腥黑穗病菌的厚垣孢子能在水中萌发，有机肥浸出液对其萌发有刺激作用。萌发适温16～20℃。病菌侵入麦苗温度5～20℃，最适9～12℃。湿润土壤（土壤持水量40%以下）有利于孢子萌发和侵染。一般播种较深，不利于麦苗出土，但增加了病菌侵染机会，病害加重发生。病菌只能侵害未出土的幼芽，而不能侵害小麦的幼苗或植株，小麦一旦出苗后，病菌就不再侵染。所以小麦腥黑穗病防治必须抓好播种期这一关键时期。在小麦腥黑穗病的发生上，一般播种愈深愈晚，出土愈慢，发病愈重。土壤温度在5～12℃、土壤湿度中等时，最容易侵染。因此，冬麦迟播，春麦早播，发病较重。4.1 小麦腥黑穗病传播途径主要是种子传播。4.2 受小麦腥黑穗病侵染的小麦，从播种到抽穗前，田间基本没有明显的症状表现，只是到小麦落黄以后症状才表现出来。4.3 当种子发芽时，病菌孢子一旦侵入小麦幼芽使小麦感病，难以用药剂进行防治。4.4 带有小麦腥黑穗病病菌的小麦有毒，人一旦食用，轻者头晕恶心，重者引起中毒。5.1 违章调运是造成小麦腥黑穗病发生的重要原因。新的种子法实施后，随着种子市场的放开，私拉乱调现象十分严重，加上调种单位检疫意识淡薄，逃漏检现象突出，检疫不严是造成小麦腥黑穗病传播的一个主要途径。5.2 近年来群众对麦播药剂拌种重视不够，白种下地是造成小麦腥黑穗病连年发生的一个主要原因。5.3 群众更换新品种不及时，自留种现象普遍。一般是购买一次种子，连种3～5年，造成病菌积累，小麦腥黑穗病逐年加重。5.4 农户之间相互串换种子是造成小麦腥黑穗病在不同乡、村之间快速扩散和严重发生的一个重要原因。在小麦品种更换上，大部分群众为了节省成本，往往不到正规的种子门店购种，而是相互之间串换，造成小麦腥黑穗病在村、户之间或村、村之大面积传播发病，基本上发病一家，传遍全村。5.5 播种晚出苗时间长是部分年份发病严重的一个重要因素。由于气候原因，部分年份小麦播种期较晚，地温低，小麦在土壤中发芽出苗时间延长，使小麦腥黑穗病病菌侵染机会增加，来年发病就重。6.1.1 加强检疫执法力度，开展产地检疫和种子抽样室内检验，严把引种、调种和种子外调关。6.1.2 小麦腥黑穗病发病重的地区（病穗率超过0.6%，含0.6%）的小麦必须销毁处理，秸秆必须进行焚烧，严禁沤肥或喂牲口。发病轻的地区（病穗率小于0.6%），要及时拔除，毁灭病株，单打单收，禁止留作种用。6.1.3 广泛宣传，积极引导群众及时更换新品种，严禁群众间相互串种。小麦腥黑穗病主要是通过种子传播，实践证明及时更换品种，是防治小麦腥黑穗病的最有效措施。一般做到年年更换新品种，经过3～5年的品种连续更换，即可将小麦腥黑穗病完全根除；购种时必须到正规的种子部门，选购经过检疫部门检疫合格的种子。6.1.4 轮作倒茬、适时早播。发生小麦腥黑穗病的田块可改种其他作物，或与油料、蔬菜等非禾本科作物进行轮作；在小麦播种时，播种不易过迟、过深，覆土不易过厚，缩短小麦出苗时间。6.2.1 药剂种子处理。用种子重量0.15%的20%的粉锈宁乳油拌种，可防治此病，还可兼防治小麦秋苗期白粉病和锈病。也可用2.5%适乐时悬浮剂按种子量的0.15%拌种，防效均较好。6.2.2 也可用1%石灰水浸种。石灰1kg加水100kg浸小麦，种子60kg，以水淹过种子10～13cm为宜。气温20℃浸3～4天，25℃时浸2天，30℃时浸1天。种子入水后禁止搅动以防破坏水面石灰膜，浸后晒干待播。6.2.3 在粪肥或土壤传染地区，除用药剂拌种外，还需采用以下方法才能收到最好的效果：每亩用纯六氯代苯0.5kg（50%的用1kg）加干细土2.5～7.5kg，拌匀制成毒土，与种子混合均匀，用耧播下；用豆饼、花生饼、芝麻饼、菜籽饼等油饼磨成粉末，每亩园22.5kg，加入10～15倍细土或土粪拌匀，和麦种同时播下；在病粪中加入人粪尿、油饼、青草等有机质，经堆积腐熟，然后施用。适时早播。注意事项：一切参与小麦收割的农事工具及车辆在离开疫情发生地块前，使用50%粉锈宁可湿性粉剂200倍液消毒处理。

水稻条纹叶枯病由灰飞虱传播的发生严重的水稻病毒病。该病2004年在浙江长兴突然发生，全县发病面积为663.3hm2，发病较重的田块丛病率达50%以上，株病率达17.6%，一般丛病率在10%左右，株病率在1%～5%，其中产量损失10%～30%为21.7hm2，损失30%以上为2.79hm2，涉及15个乡镇，个别严重田块颗粒无收。2005年水稻条纹叶枯病以较快的速度蔓延，6月中旬在长兴夹浦、洪桥、虹星桥、雉城等乡镇的单晚秧田和早播直播稻相继发病，部分严重田块株病率超过25%，7月10日左右，出现第二个症状表现高峰，移（抛）栽稻、直播稻不同程度发病，发病面积达1万hm2，其中66.67hm2损失产量20%以上。针对水稻条纹叶枯病流行的严峻形势，为了有效地控制病害暴发流行，确保水稻生产安全，从2005年起，我们对水稻条纹叶枯病及传毒媒介灰飞虱发生动态进行较为系统的监测，开展了防治技术的研究。现将调查试验结果综述如下：在前一年发病较重的田畈，选取有代表性的田块对水稻条纹叶枯的发生情况进行定期跟踪调查，以观察水稻条纹叶枯病的田间消长规律。从田间调查来看，秧田从6月上旬后期开始发病，6月15日出现第一个症状表现高峰；移栽到大田后，6月23日调查，丛病率为6%，株病率为0.89%，病情指数为0.08，至7月10日左右出现第二个症状表现高峰；以后随着发病株的枯死和分蘖的增加，丛病率和株病率都呈下降趋势，株病率下降相对较快，8月20日左右出现第3个症状表现小高峰（见图1、图2）。从田间发病调查情况看，发病程度最重在7月底，见图3。图1 水稻条纹叶枯病丛发病率增长动态（浙江长兴）图2 水稻条纹叶枯病株病发病率增长动态（浙江长兴）图3 水稻条纹叶枯病病情指数增长动态（浙江长兴）2005年灯下监测，5月中旬成虫开始上升，6月中旬出现了第2代成虫高峰，诱虫量大，诱虫量为2936头，7、8月出现了3、4代成虫的小高峰，9月出现了5代成虫高峰，9月中旬和下旬分别诱到成虫1522头和1722头，10月还有大量的成虫出现，见图4。图4 灯下灰飞虱成虫消长情况（浙江长兴，2005）2006年灯下监测，5月上旬灯下始见，下旬成虫开始上升，迁入水稻秧田为害，6月上旬后期至中旬出现了第2代成虫高峰，为全年虫量最高，诱虫达6337头，7月上旬、8月上旬又出现了3、4代成虫的小高峰，8月底至9月初后灯下虫量上升，出现了5代成虫高峰，诱到成虫1520头；10月灯下诱虫量减少，见图5。图5 灯下灰飞虱成虫消长情况（浙江长兴，2006）2005年4月22日麦田调查，越冬代虫量为5.4万头/hm2，6月16日秧田虫量为13.05万头/hm2，6月21日平均卵量480万粒/hm2，6月28日秧田虫量为4.05万头/hm2，7月下旬虫量为1.05万头/hm2，8月上中旬虫量为0.45万头/hm2，9月上旬虫量为1.5万头/hm2，9月15日虫量为4.05万头/hm2，9月20日虫量为7.5万头/hm2，9月26日虫量为16.5万头/hm2，9月30日虫量为25.5万头/hm2，10月5日虫量为33万头/hm2，10月10日虫量为27万头/hm2，10月15日虫量为10.95万头/hm2。2006年4月上旬麦田调查，越冬代虫量为15.9万头/hm2，6月30日秧田虫量为16.95万头/hm2，7月3日秧田虫量为8.55万头/hm2，7月中下旬田间虫量下降，8月1日虫量为8.55万头/hm2，8月14日虫量为19.95万头/hm2，8月25日虫量为17.25万头/hm2，8月28日虫量为20.1万头/hm2，9月8日虫量为24万头/hm2，9月下旬虫量上升，9月25日为153.45万头/hm2，9月29日虫量为28.55万头/hm2，10月虫量还比较高，10月8日虫量为48万头/hm2，10月12日虫量为31.5万头/hm2，10月17日虫量为30万头/hm2。田间灰飞虱消长曲线见图6。图6 田间灰飞虱发生消长情况（浙江长兴，2006）灰飞虱的发生量和带毒率对水稻条纹叶枯病的发生有着密切的关系，近年来，随着灰飞虱发生量增加和带毒率的提高，水稻条纹叶枯病发生面积扩大，发生程度加重。据测定，2005年长兴县灰飞虱带毒率斑点免疫快速测定为11.27%，2007年达18%。水稻灰飞虱生物法测定传毒率，2006年为6.7%，2007年为7.4%。田间观察，5月中下旬的1代灰飞虱成虫传毒造成秧田和部分早播直播田（5月下旬播种）发病，至6月中旬左右出现了水稻条纹叶枯病的第一个显症高峰；6月上旬后期至中旬的2代灰飞虱成虫高峰造成了7月中旬左右的水稻条纹叶枯病的第2个显症高峰，由于6月上旬后期至中旬的灰飞虱成虫高峰量较大，因此，7月10日左右水稻条纹叶枯病表现高峰来势凶猛，发病面积大，发病程度重；8月中旬在病情发展上有一个小高峰，如2006年8月7日左右个别失治田块出现了第3个症状表现高峰，株病率达20%以上。以后随着水稻的生长，抗逆能力增强，田间虽有大量的灰飞虱成若虫，但基本不发病。水稻条纹叶枯病的防治应立足于预防，采取“抗、避、断、治”的综合防治措施。在目前水稻对条纹叶枯病防治还没有高抗品种的情况下，重点要切断灰飞虱的传毒途径。为此，我们围绕灰飞虱的防治开展一系列的农业防治措施和药剂防治试验，根据水稻条纹叶枯病的感病期，强化药剂浸种、拌种处理和秧苗期、大田前期灰飞虱的防治，配合使用病毒钝化剂，水稻条纹叶枯病得到了有效地控制。在条纹叶枯病重发区，推广秀水63、秀水09等抗病性好的品种，压缩武运粳7号、加育991等感病品种种植。浙江长兴1代灰飞虱成虫迁移高峰期在5月中下旬，推迟至6月上旬播种的直播稻可避开大部分1代灰飞虱的迁入传毒，减少发病机率。水稻条纹叶枯病的感病期主要在秧苗期，水稻秧田期的灰飞虱防治尤其重要。因此，要及时清除农田周边杂草，5月上旬前冬闲田提前翻耕，减少灰飞虱中间寄主，恶化灰飞虱生存环境，抓好麦田灰飞虱的防治，麦田收割后及时灌水翻耕，并抓好“四边”杂草中灰飞虱的防治。同时抓好药剂浸种、拌种处理的秧田期和大田前期的灰飞虱防治。坚持“治杂草和麦田保秧田、治秧田保大田、治大田前期保大田后期”的策略。经过几年来的试验示范，防治灰飞虱以5%锐劲特45～50ml/667m2、40%毒死蜱100～120ml/667m2、50%稻丰散100ml/667m2，加水50kg喷雾防治为佳。在浸种灵浸种的基础上，催芽露白后每千克种子用35%丁硫克百威（稻拌成、稻伴）种子处理干粉剂5g拌种，混拌均匀后播种，随拌随用。在水稻发病前和发病初期，配合使用病毒钝化剂2%菌克毒克100～150ml/667m2防治1～2次，减轻水稻发病程度，减少水稻产量损失。

核盘菌（Sclerotinia sclerotiorum）所致的油菜菌核病是毁灭性的。抗病性鉴定方法是抗病材料筛选和育种的关键。本研究探讨了一种有效的田间病害圃鉴定筛选的方法，该方法中维持适中的病害压力是鉴定区别油菜品系抗性的关键。病害圃中每年连作油菜，在播种前每行施两粒菌核。在开花期利用喷雾系统喷雾保湿。于成熟期按0～4级分级调查病害。在两年的试验中，90个品系发病率在3.3%～100%。发病率和病情指数在重复之间显著相关。小区的病情指数和相对抗性指数基本为正态分布。研究结果表明该方法是有效的、有用的和灵敏的。Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape(Brassica napus).Methods for identification of resistance to S.sclerotiorum are crucial to screening and selection of resistance materials.In the study,we described a field disease nursery method efficient for resistance screening of breeding lines or germplasm of oilseed rape where maintaining of a suitable disease pressure is considered to be most important in order to differentiate levels of resistance existed in different lines.In the disease nursery,S.sclerotiorum inoculum had been maintained by growing oilseed rape consecutively and by placing two sclerotia in each row before sowing in each of the previous four seasons.During the flowering time,all plants were sprayed with water using a spray system.At maturity,disease severity was assessed on a 0~4 scale and disease index was calculated.In tests of two years,percent diseased plants of 90 lines(3 replicates in each year)ranged from 3.3%~100%.The percent of diseased plants and disease indices were significantly correlated between replicates(P<0.05).The frequency distributions of both disease indices(each plot)and relative resistance indices were in a normal form while the frequency distribution of percent diseased plants was negatively skewed.These data indicated that the method is efficient and useful to differentiate resistance of oilseed rape varieties.

核盘菌是普遍存在的坏死性真菌病原，能够侵染75科408种植物。在寄主植物中尚未发现免疫种或单基因抗性，包括十字花科。然而在禾本科等非寄主植物中该病菌是不致病的或弱致病的。为研究非寄主抗性机制，我们用该病菌接种了竹子、小麦、玉米和油菜。接种后24 h，这些植物对病菌的反应是不同的，竹子和玉米上无病斑，小麦上有小的病斑，但不同品种有变化，而油菜上产生了大的病斑。扫描电子显微镜研究表明，接种的竹子叶表面形成了一层膜，但在光学镜下菌丝穿透进入了上表皮细胞。菌丝进入竹子和油菜叶表皮细胞的方式是不同的。在油菜上菌丝很快进入上表皮细胞和细胞间隙，但竹子中菌丝仅限于上表皮和叶肉细胞。我们推定禾本科植物叶表面物质和细胞壁成分可能是阻止菌丝进入细胞的重要障碍。需要进一步分析这些成分。Sclerotinia sclerotiorium(Lib.)de Bary is a ubiquitous necrotrophic fungal pathogen capable of infecting at least 408 plant species of 75 families.No highly resistant varieties or germplasm is found in hosts including Cruciferae plants.On non-host plants such as some Gramineae species,however,the pathogen is avirulent or weak virulent.To understand how these non-hosts resist the pathogen,we inoculated S.sclerotinia mycelium to the leaves of bamboo,wheat,maize and oilseed rape as well.There were different responses in these plants after 24 hpi(hours post inoculation).No lesion was found on bamboo and maize leaves.Small lesions were observed on wheat,but the lesion size varied among different cultivars.Larger lesions were observed on oilseed rape leaves than any other Gramineaes at earlier time after inoculation.The scanning electron microscope(SEM)study showed clearly that inoculated bamboo leaf formed a layer of membrane on the leaf surface,but the slides under a light microscope unveiled hyphae penetration into the epidermal cells.The modes that hyphae grew into the leaves were also different between bamboo and oilseed rape.The hyphal growthfast under the oilseed rape epidermal and in the intercellular space,but in bamboo the growth was limited in the epidermis and mesophyll cells.We assumed that surface substance and cell wall composition are important obstacles of the hyphal penetration in non-host Gramineae plants.Further work needs to be done to analyze these compounds in comparison with oilseed rape.

Plant and fungal cells are surrounded by a cell wall rich in diverse polysaccharides and proteins.It has become apparent in recent years that the carbohydrates in the cell wall function not only to maintain cell shape and integrity,but also may serve as signals in plants(Mohnen et al.,1993).Oligogalacturonic acid(OGA),a well studied elicitor,is derived from plant cell walls(Nothnagel et al.,1983).When added to cultured plant cells,it induces an oxidative burst within minutes,releasing ROS via a pathway that involves receptor binding,activation of a G-protein,influx of Ca2+,stimulation of phospholipase C,and induction of a number of kinases(Apostol et al.,1989;Horn et al.,1989;Legendre et al.,1992;Chandra et al.,1995;Legendre et al.,1993).Purified OGAs 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro(Philippe et al.,1995).OGAs are also involved in the induction of the jasmonate pathway during plant defense response to E.carotovora subsp.Carotovora attack(Cecilia et al.,1999).The first response observed after the addition of OGAs that is clearly involved in plant defense is the production of active oxygen species,including H2O2,and O2-.This response,termed the oxidative burst,occurs within a few minutes after the addition of OGAs to suspension-cultured soybean,tobacco,and tomato cells.Reactive oxygen species are thought to have direct(through cytotoxicity)and indirect(through signaling)roles in the plant cell death required for the HR.Reactive oxygen species induce the expression of defense related genes,and are implicated as second messengers that elicit other defense responses,including systemic acquired resistance(SAR)and the HR(Brent etal.,2001).Different elicitors are thought to activate different sets of second messengers.The two signaling events that appear to participate in the OGAs inducing plant defense include the oxidative burst and NO accumulation.Inhibitors of mammalian nitric oxide synthase reduced both OGA-induced NO ac-cumulation and NOS activity,suggesting that OGA-induced NO production occurs via a NOS-like enzyme(Hu et al.,2003). Nitric oxide(NO)is a highly reactive molecule that rapidly diffuses and permeates cell membranes.During the last few years NO has a significant role in plant resist-ance to pathogens by triggering resistance-associated cell death and by contributing to the local and systemic induction of defense genes.NO stimulates signal transduction pathways through protein ki-nases,cytosolic Ca2+mobilization and protein modification(María et al.,2004). Most of the ex-perimental data available on NO detection during plant-pathogen interactions come from studies of infections by biotrophic pathogens(María et al.,2004). Additionally,an increase in NOS activity correlated with the pathogen resistance response has been observed in resistant tobacco during TMV infection( Durner et al.,1998;Chandok et al.,2003).Here we report that OGAs induced a range of defense responses in tobacco,including oxidative burst,NO accumulation and stimulation of superoxide dismutase(SOD)activity and catalase(CAT)activity.Furthermore,we show that tobacco plant sprayed with OGAs developed a resistance against infection by tobacco mosaic virus.We also provide evidence that the defense response induced by OGAs was connected with H2O2 and NO pathway.Plants of tobacco(Nicotiana tabacum var.sam sun NN)were grown from seeds in a greenhouse and were used at the 4~6-leaf stage after 2 months in culture.The plants were kept in a growth chamber at(23±1)℃ with a photoperiod of 16 h and 70%~80%relative humidity for several days before treatments.Diphenylene iodonium(DPI),2-(N-morpholino)ethanesulfonic acid(MES),Sodium nitroprusside(SNP),catalase(CAT,from bovine liver),NG-nitro-L-arginine-methyl eater(L-NAME)and 4,5-diaminofluorescein diacetate(DAF-2 DA)were obtained from Sigma.2′,7′-dichlorofluorescin diacetate(H2DCF-DA)from Biotium.All other reagents were from Shanghai Chemical Reagent CO.,LTD,Tianjin Kermel Chemical Development Centre,or Beijing Chemical Plant.OGAs was prepared from enzymatic hydrolysis of pectin and separated with membrane according to the report(H Zhang et al.,1999).An aliquot of OGAs was dissolved in water and analyzed with a matrix-assisted laser desorption-ionization time-of-flight mass spectrometer(MALID-TOF-MS,Bruker,Germany).Tobacco mosaic virus(TMV)that came from our collection was multiplied in N.tabacum.TMV was extracted from systemic infected plants by homogenization of infected leaves in 0.05mol/LH3PO4 buffer(0.05mol/L KH2PO4,0.05 M Na2HPO4 pH 6.8)with subsequent clarification of the extract by centrifugation at 2000g for 6 min.The supernatant extract was used for mechanical inoculation.All leaves of plant were sprayed with 50μg/ml of OGAs,the control plants were sprayed with water.24h~25d after OGAs application,plants were inoculated mechanically with TMV.The lesion caused by TMV was investigated at 7d after inoculation.Results were analyzed using Duncan's multiple range test at P= 0.05.For measurements of SOD and CAT activities,tobacco leaves treated with OGAs were kept in liquid nitrogen.The enzymes in the frozen powders were extracted by adding 0.05g polyvinylpyrrolidone and 5ml 0.05mol/L sodium borate buffer at pH 8.8 and homogenized at 4℃.SOD activities were measured as described by Zhu Guanglian(Zhu Guanglian et al.,1990).CAT activity was determined using the method of Beers&Sizers(Beer et al.,1952).NO and H2O2 measurement was performed using their fluorescent indicator dye DAF-2 DA and H2DCF-DA as described previously by H.Kojima(H.Kojima et al.,1998)with slight modifications.The epidermis was peeled carefully from abaxial surface of the leaves and cut into 5-mm length.Epidermal strips were placed into Tris/KCl buffer(Tris 10 mmol/L and KCl 50mmol/L,pH 7.2)containing DAF-2 DA at a final concentration of 10μmol/L for 30min,or H2DCF-DA at 50μmol/L for 10min,at 26℃ in the dark.The epidermal sections were removed and transferred to a dish of fresh Tris/KCl buffer(without probe)to wash off excess fluorophore apart from light.Then the epidermal strips were placed in Tris/KCl buffer containing OGAs and inhibitors.Examination of peels was performed using laser scanning confocal microscopy(Leica,TCS SP2)with exciting wavelength 488 nm,emitting wavelength 505~530nm.Plants were sprayed with 0.01 and 0.1 mmol/L of sodium nitroprusside(SNP),50μg/ml of OGAs,1 mmol/L,10mmol/L and 100 mmol/L H2O2,H2O2 scavenger catalase(CAT,100unit/ml)and OGAs cotreatment,H2O2 scavenger ascorbic acid(0.1mmol/L)and OGAs cotreatment and NOS inhibitor L-NAME(1mmol/L)for 30min before OGAs respectively.The control plants were sprayed with water.In all cases,24h after OGAs and other materials applications,plants were inoculated with TMV.The lesion caused by TMV was investigated at 7d after inoculation.The effect of OGAs,SNP and H2O2 on local infection was calculated from the ratio of the number of local lesion produced on the treated leaves to that on the control leaves treated with water.The TOF-MS profiles of OGAs sample were showed in Figure 1.The mass spectrum indicated that peaks corresponding to the mass numbers of( M+ Na)+of trimer to enneamer were detected.So the sample was composed mainly of OGAs having degree of polymerization( DP)2-8.Figure 1 TOF-MS of oligochitosan sampleThe results of control effects on TMV with OGAs at different concentration(50～100μg/ml)showed that the best concentration was 50 μg/ml(data not shown).The effects of application of OGAs at different time were summarized in Table 1.It was found that tobacco leaves treated with OGAs were protected against TMV infection.When the inoculation occurred at 19d after spraying 50μg/ml OGAs on tobacco plants,the relative control effect was 53.42%.We concluded that the resistance induced by OGAs became better with the inducing time until 19d.The resistance was reduced after 19d.Dayof50μg/mlgalacturonideappliedNumberoflesioncausedbyTMVRelativecontroleffect(%)vcdsaw1d125±5814.40a?4d116±3920.55a?7d120±4217.81a10d84±3742.47ab13d97±4433.56ab16d90±3238.36ab19d68±3453.42b22d71±3451.37b25d89±3739.04bck146±51—Table 1We examined the effects of OGAs on the activity of plant resistance correlated enzymes.The results(Figure 2 and Figure 3.)indicated that OGAs increased activity of SOD and CAT compared with the H2O-treated ones.There are no distinct differences on the activity of POD and PPO of tobacco leaves treated with OGAs or water(data not shown).SOD and CAT are concerned with eliminating oxygen free radical.Within one hour,activities of CAT and SOD were induced to maximum.Figure 2 Time course of SOD activity in tobacco leaves treated by 50μg/ml OGAs or H2O as CKFigure 3 Time course of CAT activity in tobacco leaves treated by 50μg/ml OGAs or H2O as CKBecause of activity of SOD and CAT induced by OGAs and the two enzymes correlative with oxygen free radical,we examined the production of H2O2 induced by OGAs.To study the effects of OGAs on the production of H2O2 in tobacco cells,the H2O2-sensitive fluorophore H2DCF-DA were used.The results of production of H2O2 in epidermal cells of tobacco leaves induced by OGAs were shown in Figure 4.It was found that OGAs caused an increase of intracellular H2DCF-DA fluorescence in epidermal cells and guard cells of tobacco leaves,indicating the production of H2O2.Fluorescence became visible along the plasma membrane and in organelles in the epidermal cells of tobacco leaves treated with OGAs(Figure 4C),but the fluorescence was very faint in the epidermal cells only loaded with H2DCF-DA(Figure 4A).The Figure 4E and G showed that CAT and DPI could inhibit the level of H2DCF-DA fluorescence in the cells of tobacco leaves treated with OGAs.The results revealed that CAT and DPI could suppress the production of H2O2.Figure 4 Laser scanning confocal microscopy of OGA-induced production of H2O2 in epidermal cells of tobacco leaves. (A) The cells loaded with H2DCF-DA. (B) Bright field image of the cells loaded with H2DCF-DA. (C) The cells loaded with H2DCF-DA before treatment with OGA. (D)Bright field image of the cells loaded with H2DCF-DA before treatment with OGA. (E) The cells loaded with H2DCF-DA and elicited by OGA in the presence of the CAT. (F) Bright field image of the cells loaded with H2DCF-DA and elicited by OGA in the presence of the CAT. (G) The cells loaded with H2DCF-DA and elicited by OGA in the presence of the DPI. (H) Bright field image of the cells loaded with H2DCF-DA and elicited by OGA in the presence of the DPI.The NO-sensitive fluorophore DAF-2DA was used to observe NO accumulation.The observed LSCM results of OGAs-induced production of NO in epidermal cells of tobacco leaves were shown in Figure 5.It was found that OGAs could enhance the level of intracellular DAF-2DA fluorescence in epidermal cells of tobacco leaves,indicating massive production of NO.Production of NO and/or accumulation was observed in organelles and along the plasma membrane in the epidermal cells of tobacco leaves treated with OGAs(Figure 5C).However,the DAF-2DA fluorescence indicating production of NO was not observed in the epidermal cells only loaded with DAF-2DA(Figure 5A).The results also indicated that CPTIO and L-NAME could inhibit the level of H2DCF-DA fluorescence in the cells of tobacco leaves treated with OGAs(Figure 5E and G).The results representedthat CPTIO and L-NAME could suppress the production of NO.Figure 5 Laser scanning confocal microscopy of OGA-induced production of NO in epidermal cells of tobacco leaves. (A) The cells loaded with DAF-2 DA. (B) Bright field image of the cells loaded with DAF-2 DA. (C) The cells loaded with DAF-2 DA before treatment with OGA. (D) Bright field image of the cells loaded with DAF-2 DA before treatment with OGA. (E) The cells loaded with DAF-2DA and elicited by OGA in the presence of the CPTIO. (F) Bright field image of the cells loaded with DAF-2DA and elicited by OGA in the presence of the CPTIO. (G) The cells loaded with DAF-2DA and elicited by OGA in the presence of the L-NAME. (H) Bright field image of the cells loaded with DAF-2DA and elicited by OGA in the presence of the L-NAME.As H2O2 and NO appear to be a key factor associated with plant induced defense disease,it was interesting to test the effect of exogenous NO and H2O2.The effect of OGAs,NO donor SNP and H2O2 at different concentrations and some scavengers are summarized in Figure 6.It was found that treatment with OGAs,SNP and H2O2 protected tobacco leaves against TMV local infection.The least lesion was observed at the treatment of 50μg/ml OGAs among the all treatments.The inhibition effect of H2O2 showed dependence on the amount of H2O2.The lesion of co-treatment of OGAs and the H2O2 scavenger CAT or ascorbic acid on TMV infection was as high as CK.We also observed SNP inducing resistance was dose-dependent.When the tobacco plants were treated with L-NAME before OGAs,the induced resistance was depressed.Therefore,we can presume NO and H2O2 are important factors participating in OGAs inducing resistance to TMV.Figure 6 Effect of OGAs and exogenous NO and H2O2 on disease symptomPectic oligosaccharides,produced by microbial enzymes,are well-known oligosaccharins,eliciting defence responses in diseased plants(Dumville et al.,2000).A broad spectrum of OG-induced pathogenesis-related defense responses has been reported(M.T.Esquerré-Tugayé et al.,2000).Most defense and developmental responses are induced by OGAs with a degree of polymerization(DP)between 10 and 15 galacturonic acid residues.OGAs with a DP less than 8 can also trigger defense responses in plants:they induce accumulation of protease inhibitors(T.Moloshok et al.,1992),ethylene production(S.D.Simpson et al.,1998)and elicitation of genes involved in jasmonic acid metabolism in tomato(C.Norman et al.,1999).In this report,we observed the OGAs with a DP between 2~8 could induce tobacco resistance to TMV.The concentration of OGAs used was also discussed.OGAs-induced plant growth has been reported(LoSchiavo et al.,1991;Filippini et al.,1992),and the maximal effect to growth was about 10-4 M(Stephen et al.,1993).To elicit plant defense responses,OGAs concentration higher than those usually required for control developmental process.In our experiments,50μg/ml was the best concentration to induce resistance within 100μg/ml(data not shown).It showed the efficiency of the OGAs in inhibition of virus infection was not depended on the dose of OGAs.But the inhibition effect was dependent on the treatment time.We observed the inducing effect of resistance to TMV was gradually elevated before 19d,but the mechanism of this needed further study.Research showed that lag period of the induced resistance of glucohexaose was about 7days and the protection period was about 28 days(Li Hongxia et al.,2005).Furthermore,tobacco plants treated by sulfated fucan or linear β-1,3 glucan showed resistance to TMV or bacterium E.carotovora after 5 days(Olivier Klarzynski et al.,2003;2000).So far no oligosaccharides were reported to have so long time inducing effect.Therefore,OGAs have more predominance to be applied in agriculture.Experimental results also showed that NO and H2O2 played important roles in OGAs inducing tobacco resistance to TMV.NO and H2O2 as important signaling active molecules in pathogen defense reaction has been extensively studied(Levine et al.,1994;Mehdy et al.,1996;Baker et al.,1995;Jabs et al.,1996;Delledonne et al.,1998;Rout-Mayer et al.,1997?;Binet et al.,1998).First,we examine the activity of plant resistance correlated enzymes.Because the activity of PAL has been confirmed elevated by many reports(Messiaen et al.,1994;Lapous et al.,1998;Dixon et al.,1989;Tepper et al.,1990),we just mensurated the PPO,POD,SOD and CAT.This includes the activity of SOD and CAT elevated,so we estimated the extra H2O2 production.To evaluate the stimulatory effect of OGAs on tobacco cells,we measured the production of H2O2 and NO in tobacco cells.The data indicated that OGAs induced the production of H2O2 and NO in epidermal cells of tobacco within a short time.These results were in agreement with the reports by Xiangyang Hu,who claimed OGAs stimulated NO accumulation in the growth medium of ginseng suspension cultures(Hu et al.,2003).Rout-Mayer and Binet discovered respectively H2O2 production within a few minutes after the addition of OGAs to suspension-cultured tobacco cells(Rout-Mayer et al.,1997;Binet et al.,1998).Many reports show H2O2 and NO exist are correlated to plant defense.H2O2 is involved in the induction and/or execution of hypersensitive reaction(C.S.Bestwick et al.,1997).H2O2 is required for the cross-linking of plant cell wall components as a part of the structural defense response(C.Lamb et al.,1997).The production of H2O2 may also lead to the development of an antimicrobial environment within the apoplast(M.Peng et al.,1992).In many cases,H2O2 collaborate with NO to execute invading pathogens.H2O2 and NO production were induced almost at the same time by cryptogein,a fungal elicitor(Foissner et al.,2000).NOS inhibitors compromise the hypersensitive resistance response in Arabidposis and tobacco(Delledonne et al.,1998?;Huang et al.,1998).TMV infection could elevate NOS(nitric oxide synthase)activity,and NO could induce PR-1 expression(Durner et al.,1998).NO,as well as other ROS,have been shown to stimulate the accumulation of SA(Durner et al.,1999),which play a critical signaling role in the activation of plant defense responses after pathogen attack.Furthermore,to test whether OGAs functions on inducing resistance in tobacco via NO and H2O2 pathway,we examined the effects of OGAs,exogenous NO donor SNP and H2O2 on inducing resistance to TMV.It was found that all of these treatments reduced lesion caused by TMV.But co-treatment with OGAs and H2O2 scavenger CAT or ascorbic acid blocked the inducing resistance.The tobacco plants inhibited NOS activity by L-NAME were not induced resistance by OGAs.So the defense response induced by OGAs was connected with NO and H2O2 pathway.The study reported herein reveals that OGAs can induce the production of H2O2 and NO,and induce the defense response against TMV.Our understanding of OGAs induced resistance is sketchy.The mechanisms of OGAs eliciting defense responses of tobacco need further investigation.