馬鈴薯主栽品種抗馬鈴薯金線蟲(chóng)鑒定及抗性分子標(biāo)記檢測(cè)

黃立強(qiáng)，江如，朱波汁，彭煥，許翀，宋家雄，陳敏，李永青，黃文坤，彭德良

馬鈴薯主栽品種抗馬鈴薯金線蟲(chóng)鑒定及抗性分子標(biāo)記檢測(cè)

黃立強(qiáng)1，江如1，朱波汁1，彭煥1，許翀2，宋家雄2，陳敏2，李永青2，黃文坤1，彭德良1

1中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所植物病蟲(chóng)害綜合治理全國(guó)重點(diǎn)實(shí)驗(yàn)室，北京 100193；2云南省昭通市植保植檢站，云南昭通 657000

【目的】馬鈴薯金線蟲(chóng)（）是國(guó)際公認(rèn)的重要檢疫性有害生物，目前已在云南、貴州、四川3省7縣（市）發(fā)生危害，產(chǎn)區(qū)內(nèi)多個(gè)種薯基地受到傳播威脅。通過(guò)西南地區(qū)馬鈴薯主栽品種對(duì)馬鈴薯金線蟲(chóng)的抗性鑒定、分子標(biāo)記檢測(cè)及田間抗性評(píng)價(jià)，明確已知抗病基因的分布情況，為該地區(qū)馬鈴薯金線蟲(chóng)應(yīng)急防控、抗病品種合理布局和良種推廣提供依據(jù)?！痉椒ā坷迷颇像R鈴薯金線蟲(chóng)群體對(duì)15份馬鈴薯主栽品種進(jìn)行室內(nèi)盆栽接種，計(jì)算最終單株孢囊數(shù)和相對(duì)感病性，根據(jù)抗性等級(jí)劃分標(biāo)準(zhǔn)進(jìn)行抗性評(píng)價(jià)；同時(shí)利用57R和TG689分子標(biāo)記鑒定抗馬鈴薯金線蟲(chóng)，以-胡蘿卜素羥化酶基因的BCH分子標(biāo)記為對(duì)照；并于2020年和2021年在云南省昭通市開(kāi)展兩年度的田間試驗(yàn)，在播種前和收獲后分別采集土壤樣品并分離孢囊，計(jì)算播種前初始群體密度（Pi）、最終群體密度（Pf）及平均繁殖系數(shù)（Pf/Pi）。馬鈴薯現(xiàn)蕾至始花期測(cè)定株高，收獲時(shí)測(cè)定產(chǎn)量。【結(jié)果】15個(gè)馬鈴薯主栽品種中的云薯505、宣薯5號(hào)、會(huì)薯15號(hào)、會(huì)薯19號(hào)以及云薯304共5個(gè)品種為高抗品種，馬鈴薯金線蟲(chóng)基本不能在這些品種上繁殖；麗薯6號(hào)、宣薯6號(hào)為中感品種；其余8個(gè)為高感品種，尤其是會(huì)-2、麗薯15號(hào)以及宣薯8號(hào)的平均繁殖系數(shù)高于感病對(duì)照品種會(huì)薯16號(hào)（Pf/Pi=17.15）。兩個(gè)基因鑒定分子標(biāo)記結(jié)果大致相同，5個(gè)馬鈴薯品種，即云薯505、宣薯5號(hào)、會(huì)薯15號(hào)、云薯304和宣薯6號(hào)含。馬鈴薯金線蟲(chóng)的繁殖系數(shù)在田間抗/感馬鈴薯品種上具有顯著性差異，抗性等級(jí)為9的高抗品種田間平均繁殖系數(shù)在2021年（0.04—0.12）和2022年（0.05—0.14）均＜1.00，表明高抗品種種植后線蟲(chóng)田間群體密度有一定程度的降低。高感品種平均繁殖系數(shù)在2021年（1.18—2.75）和2022年（1.76—3.24）均＞1.00，高感品種種植后田間線蟲(chóng)種群數(shù)量增加。不同品種間株高和產(chǎn)量差異顯著（＜0.05），5個(gè)高抗品種株高的均值在兩年間均顯著高于8個(gè)高感品種。宣薯5號(hào)、會(huì)薯15號(hào)和會(huì)薯19號(hào)產(chǎn)量最高，兩年度分別為51.67—56.48和33.28—40.57 t·hm-2，會(huì)-2產(chǎn)量最低?！窘Y(jié)論】西南混作區(qū)主栽馬鈴薯高抗品種對(duì)馬鈴薯金線蟲(chóng)具有優(yōu)良抗性，主要攜帶抗病基因且能夠顯著減少田間馬鈴薯金線蟲(chóng)群體密度。高抗品種中宣薯5號(hào)、會(huì)薯15號(hào)和會(huì)薯19號(hào)為高產(chǎn)抗病良種，云薯304為富鋅薯片加工型品種。亟待根據(jù)馬鈴薯金線蟲(chóng)的發(fā)生分布，進(jìn)一步有針對(duì)性地?cái)U(kuò)大對(duì)主栽品種抗性水平的鑒定。

馬鈴薯金線蟲(chóng)；馬鈴薯抗性品種；抗病基因；分子標(biāo)記檢測(cè)

0 引言

【研究意義】馬鈴薯（）作為我國(guó)的第四大糧食作物，其種植面積和產(chǎn)量均居世界第一，在全國(guó)范圍內(nèi)均有種植，尤其是在貧困地區(qū)種植面積較大，在保障糧食安全和脫貧攻堅(jiān)戰(zhàn)中發(fā)揮了重要作用。馬鈴薯金線蟲(chóng)（）是國(guó)際公認(rèn)的重要檢疫性有害生物，嚴(yán)重危害馬鈴薯，寄生番茄、茄子等126種茄科植物[1-3]。2018年我國(guó)在貴州省首次發(fā)現(xiàn)馬鈴薯金線蟲(chóng)，目前已在云南、貴州、四川3省7縣（市）發(fā)現(xiàn)該線蟲(chóng)的發(fā)生危害[4-7]。種植抗病品種仍然是其最具經(jīng)濟(jì)效益和生態(tài)效益的防治方法，篩選可直接用于馬鈴薯金線蟲(chóng)防控的現(xiàn)有主栽馬鈴薯品種對(duì)有效遏制金線蟲(chóng)的擴(kuò)散蔓延具有重要意義，可為我國(guó)馬鈴薯金線蟲(chóng)應(yīng)急防控和可持續(xù)防控技術(shù)提供重要科學(xué)依據(jù)?！厩叭搜芯窟M(jìn)展】馬鈴薯金線蟲(chóng)發(fā)生后一般對(duì)馬鈴薯造成20%—30%的產(chǎn)量損失，發(fā)病嚴(yán)重時(shí)損失可高達(dá)80%—90%，甚至絕收[8]。該線蟲(chóng)屬固著性內(nèi)寄生線蟲(chóng)，以鞣質(zhì)孢囊在土壤內(nèi)越冬、滯育。由于孢囊擁有極強(qiáng)抵抗不良環(huán)境和殺線蟲(chóng)劑的能力，這也成為該線蟲(chóng)難以被根除的主要原因。馬鈴薯金線蟲(chóng)已在六大洲超過(guò)100個(gè)國(guó)家發(fā)生[3,9]。由于其危害嚴(yán)重，美國(guó)、歐盟、歐洲和地中海植物保護(hù)組織（EPPO）和中國(guó)在內(nèi)的全球106個(gè)國(guó)家和組織將其列為進(jìn)境植物檢疫性有害生物[1,3,10]。2020年，我國(guó)已將馬鈴薯金線蟲(chóng)納入《全國(guó)農(nóng)業(yè)植物檢疫性有害生物名單》。馬鈴薯金線蟲(chóng)的傳播方式分為近距離和遠(yuǎn)距離傳播，前者以2齡幼蟲(chóng)在土壤中做10—50 cm的短距離移動(dòng)，而后者可經(jīng)由風(fēng)、雨水、家畜和野生動(dòng)物、馬鈴薯種薯或商品、加工用塊莖及農(nóng)事操作、農(nóng)具和交通工具等粘帶孢囊而傳播[11-12]。前人利用具有不同抗性位點(diǎn)的一套馬鈴薯鑒別寄主可將馬鈴薯金線蟲(chóng)劃分為Ro1—Ro5 5種致病型[13]。Ro1致病型是全世界馬鈴薯產(chǎn)區(qū)分布最為廣泛的致病型，而是位于馬鈴薯野生種ssp.5號(hào)染色體上的一個(gè)顯性基因，其對(duì)馬鈴薯金線蟲(chóng)Ro1型具有優(yōu)異抗性[14-16]。目前，種植抗病品種仍然是防治該線蟲(chóng)最經(jīng)濟(jì)有效的措施。長(zhǎng)期以來(lái)，科研人員在馬鈴薯種質(zhì)資源篩選、抗病基因發(fā)掘與鑒定和育種應(yīng)用方面開(kāi)展了大量的研究工作[14,17-21]。Park等[22]通過(guò)57R和TG689分子標(biāo)記及盆栽接種對(duì)38個(gè)馬鈴薯品種和350份育種材料進(jìn)行鑒定比較，評(píng)價(jià)認(rèn)為這兩種分子標(biāo)記抗性鑒定能力均較高，分別為99.7%和98.3%，任何一個(gè)標(biāo)記都可以滿足抗病育種中對(duì)馬鈴薯金線蟲(chóng)抗性鑒定的需求；de Boer等[23]比較了馬鈴薯抗/感品種在田間對(duì)澳大利亞馬鈴薯金線蟲(chóng)群體繁殖系數(shù)的影響，證實(shí)攜帶的馬鈴薯抗性品種可以有效減少馬鈴薯金線蟲(chóng)Ro1型的種群數(shù)量；明會(huì)等[24]利用6個(gè)抗馬鈴薯金線蟲(chóng)相關(guān)分子標(biāo)記對(duì)我國(guó)875份馬鈴薯種質(zhì)資源進(jìn)行抗性鑒定，篩選得到211份馬鈴薯種質(zhì)含有抗馬鈴薯金線蟲(chóng)的?！颈狙芯壳腥朦c(diǎn)】馬鈴薯金線蟲(chóng)目前在我國(guó)云南、貴州、四川3省發(fā)生危害，產(chǎn)區(qū)內(nèi)多個(gè)種薯基地受到傳播威脅，為該線蟲(chóng)的防治工作帶來(lái)嚴(yán)峻挑戰(zhàn)。而現(xiàn)今我國(guó)西南混作區(qū)主栽馬鈴薯品種對(duì)該線蟲(chóng)的抗性水平仍不明確，品種田間抗/感表現(xiàn)及其對(duì)線蟲(chóng)群體的影響尚不清楚，亟待篩選優(yōu)質(zhì)、高抗品種以滿足馬鈴薯金線蟲(chóng)綜合防控技術(shù)措施需求?！緮M解決的關(guān)鍵問(wèn)題】通過(guò)對(duì)15份馬鈴薯西南混作區(qū)主栽馬鈴薯品種進(jìn)行盆栽接種及抗性評(píng)價(jià)并結(jié)合抗馬鈴薯金線蟲(chóng)基因分子標(biāo)記鑒定，比較抗/感品種的田間表現(xiàn)，分析主栽品種對(duì)馬鈴薯金線蟲(chóng)的抗性水平，為該地區(qū)馬鈴薯金線蟲(chóng)應(yīng)急防控形勢(shì)下馬鈴薯品種合理布局和良種推廣提供參考。

1 材料與方法

1.1 材料

供試馬鈴薯金線蟲(chóng)群體采自我國(guó)云南省昭通市的馬鈴薯根系及根際土壤，所有樣品保存于中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所植物病蟲(chóng)害綜合治理全國(guó)重點(diǎn)實(shí)驗(yàn)室。量取200 mL充分混勻的根際土壤樣品，利用淺盤(pán)法或淘洗過(guò)篩法分離孢囊、雌蟲(chóng)及2齡幼蟲(chóng)[25-26]。孢囊保存于1.5 mL離心管中，4 ℃留存?zhèn)溆谩?/p>

供試馬鈴薯為馬鈴薯西南混作區(qū)部分主栽品種，共15份。感病對(duì)照品種為會(huì)薯16號(hào)。本試驗(yàn)完成后所有材料均經(jīng)高溫滅菌處理。

1.2 室內(nèi)盆栽接種

1.2.1 馬鈴薯育苗與移栽 盆栽接種鑒定于2019年5—8月在中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所植物病蟲(chóng)害綜合治理全國(guó)重點(diǎn)實(shí)驗(yàn)室P3隔離溫室進(jìn)行。試驗(yàn)方法參照EPPO PM 7/40 (5)馬鈴薯金線蟲(chóng)和馬鈴薯白線蟲(chóng)（）診斷技術(shù)標(biāo)準(zhǔn)以及PM 3/68 (1)馬鈴薯品種對(duì)馬鈴薯孢囊線蟲(chóng)抗性評(píng)價(jià)鑒定標(biāo)準(zhǔn)[27-28]。種薯用1%次氯酸鈉溶液浸泡消毒30 min，無(wú)菌水沖洗3—5次。切下以馬鈴薯單芽眼為中心約3 cm3的薯塊于濕沙中育苗，芽長(zhǎng)至3—5 cm后，移栽至含有800 mL滅菌土（沙﹕土= 2﹕1）的花盆中。每個(gè)品種設(shè)置4個(gè)技術(shù)重復(fù)和2個(gè)生物學(xué)重復(fù)。

1.2.2 馬鈴薯金線蟲(chóng)卵懸浮液制備及接種 挑取飽滿深棕色孢囊置于100目篩網(wǎng)上，下方放置600目篩網(wǎng)。用試管塞在篩網(wǎng)上輕輕研磨孢囊，并用流水將卵沖洗到下方篩網(wǎng)中，最后將卵收集到燒杯中制備成1 000粒卵/mL的卵懸浮液。

馬鈴薯苗移栽生長(zhǎng)一周后，用竹簽在花盆內(nèi)戳4個(gè)5 cm深度的孔洞。取馬鈴薯金線蟲(chóng)卵懸浮液進(jìn)行接種，每盆接種4 mL卵懸浮液。初始群體密度（Pi）為5粒卵/mL土，每盆約接種13個(gè)孢囊。接種后，置于隔離溫室內(nèi)培養(yǎng)，培養(yǎng)條件為溫度16—22 ℃，白天光照16 h，夜晚黑暗8 h，每3 d澆水200 mL，2周澆一次N-P-K 20-20-20水溶營(yíng)養(yǎng)液。

1.2.3 室內(nèi)盆栽接種抗性評(píng)價(jià) 接種15周后，分離并計(jì)數(shù)單株孢囊數(shù)。以會(huì)薯16號(hào)作為感病對(duì)照品種，計(jì)算供試馬鈴薯品種對(duì)感病對(duì)照品種的相對(duì)感病性。相對(duì)感病性（%）=（Pf供試馬鈴薯品種/Pf感病對(duì)照品種）×100，Pf（The final population）代表最終孢囊數(shù)量。根據(jù)供試馬鈴薯品種Pf與感病對(duì)照品種Pf的相對(duì)感病性進(jìn)行抗性評(píng)價(jià)。馬鈴薯品種對(duì)馬鈴薯金線蟲(chóng)的抗性等級(jí)劃分標(biāo)準(zhǔn)見(jiàn)表1[28]。

表1 EPPO馬鈴薯品種對(duì)馬鈴薯金線蟲(chóng)抗性等級(jí)劃分標(biāo)準(zhǔn)

抗性等級(jí)分為1—9，其中抗性等級(jí)為9代表最高抗性，馬鈴薯金線蟲(chóng)基本不能侵染該品種或侵染后不能完成整個(gè)生活史

Resistance scores range from 1 to 9, where 9 represents the highest resistance level andis basically unable to infest the cultivar or cannot complete its entire life cycle after infestation

1.3 田間自然小區(qū)抗性鑒定

1.3.1 試驗(yàn)地點(diǎn)和田間管理 試驗(yàn)于2021和2022連續(xù)兩年在云南省昭通市昭陽(yáng)區(qū)西魁坪子馬鈴薯種植基地進(jìn)行（E103°41′47′′，N27°35′45′′，海拔2 270 m）。昭陽(yáng)區(qū)西魁坪子地處云南省東北部烏蒙山區(qū)腹地，地勢(shì)西高東低。海拔1 700 m以上區(qū)域?yàn)轳R鈴薯主產(chǎn)區(qū)，屬高原大陸季風(fēng)性氣候，干濕兩季分明，冬季氣溫較低，夏季氣候涼爽。年平均氣溫12.1 ℃，年降水量826 mm，年日照時(shí)數(shù)1 719.4 h，全年無(wú)霜期220 d左右，土壤類型為山地黃壤[29-30]。該試驗(yàn)基地冬春干旱，夏秋多雨，常年連作馬鈴薯，試驗(yàn)前經(jīng)調(diào)查馬鈴薯金線蟲(chóng)已侵染定殖[6]。

試驗(yàn)采用隨機(jī)區(qū)組排列，共設(shè)15個(gè)處理（品種），小區(qū)長(zhǎng)5.5 m，寬2.4 m，面積13.2 m2，小區(qū)間隔0.5 m，每處理重復(fù)3次，共45個(gè)小區(qū)。每小區(qū)起兩壟，壟高20—30 cm，采用雙行栽培模式。將N-P2O5-K2O比例為16﹕6﹕26的硫酸鉀型復(fù)合肥與精制有機(jī)肥按1﹕2充分混合后，每穴施150 g，覆土蓋膜。馬鈴薯于每年3月中旬種植，栽培管理按當(dāng)?shù)卮筇锷a(chǎn)常規(guī)措施進(jìn)行，田間無(wú)其他病蟲(chóng)害發(fā)生。在馬鈴薯現(xiàn)蕾至始花期，測(cè)量株高。每年10月中旬收獲并測(cè)產(chǎn)。

1.3.2 田間抗性測(cè)定 在馬鈴薯播種前，每小區(qū)利用網(wǎng)格土壤采樣法[31]采集亞樣品20個(gè)，取樣深度為10—20 cm，充分混合后每小區(qū)留2 kg土壤作為一個(gè)樣品帶回實(shí)驗(yàn)室進(jìn)行孢囊數(shù)測(cè)定。從每個(gè)小區(qū)樣品中取100 mL土樣，經(jīng)篩淘法分離樣品中的孢囊，在顯微鏡下觀察并統(tǒng)計(jì)播種前每個(gè)小區(qū)線蟲(chóng)孢囊基數(shù)（Pi）。在一個(gè)生長(zhǎng)季節(jié)結(jié)束，馬鈴薯收獲后，用同樣的方法從每小區(qū)取土壤樣品帶回室內(nèi)測(cè)定單位土壤內(nèi)的最終孢囊數(shù)（Pf）。計(jì)算馬鈴薯金線蟲(chóng)的繁殖系數(shù)，繁殖系數(shù)=成熟期孢囊數(shù)Pf/播種前土壤孢囊數(shù)Pi。孢囊繁殖系數(shù)（Pf/Pi）＜1，則該品種為抗病品種，反之則為感病品種。

1.4 抗馬鈴薯金線蟲(chóng)H1基因鑒定

馬鈴薯出苗后2周取頂部嫩葉于液氮速凍，使用CTAB法提取植物葉片DNA[32]?；蚪MDNA用Nanodrop 2000分光光度計(jì)（Thermo Fisher Scientific Inc.，Wlimington，DE）測(cè)定DNA濃度及純度，并用滅菌水將DNA稀釋至100 ng·mL-1左右，樣品保存于-80 ℃冰箱。采用57R[17]和TG689[33]兩個(gè)分子標(biāo)記鑒定抗馬鈴薯金線蟲(chóng)Ro1致病型的，分子標(biāo)記BCH[22]擴(kuò)增-胡蘿卜素羥化酶基因作為對(duì)照。對(duì)于57R分子標(biāo)記，抗馬鈴薯金線蟲(chóng)品種擴(kuò)增得到450 bp的條帶，感病品種擴(kuò)增出1 500 bp的條帶，而前人研究認(rèn)為同時(shí)出現(xiàn)1 500和450 bp條帶的馬鈴薯品種也同樣抗馬鈴薯金線蟲(chóng)，因此也認(rèn)定其含有[22]。TG689分子標(biāo)記則相對(duì)特異，抗性品種擴(kuò)增出141 bp大小的條帶，而感病品種無(wú)條帶。分子標(biāo)記擴(kuò)增引物序列如表2，以上所有引物均由上海生工生物工程股份有限公司合成。PCR反應(yīng)體系25 μL：12.5 μL 2×Rapid Taq Master Mix（南京諾唯贊生物科技股份有限公司），0.5 μL 10 μmol·L-1正/反引物，1 μL DNA模板，滅菌水補(bǔ)足至25 μL。PCR程序：94 ℃預(yù)變性3 min；94 ℃變性15 s，55 ℃退火15 s，72 ℃延伸15 s，30個(gè)循環(huán)；72 ℃延伸10 min，4 ℃保存。取10 μL擴(kuò)增產(chǎn)物經(jīng)1.2%瓊脂糖凝膠電泳分離，全自動(dòng)凝膠成像分析系統(tǒng)（北京森西賽智科技有限公司）下觀察并記錄結(jié)果。

1.5 數(shù)據(jù)分析

應(yīng)用Microsoft Excel和IBM SPSS Statistics 22數(shù)據(jù)處理軟件進(jìn)行統(tǒng)計(jì)分析，采用Duncan新復(fù)極差法對(duì)不同品種間產(chǎn)量進(jìn)行多重比較。

表2 抗馬鈴薯金線蟲(chóng)H1基因分子標(biāo)記鑒定

2 結(jié)果

2.1 馬鈴薯抗馬鈴薯金線蟲(chóng)室內(nèi)接種鑒定

對(duì)15個(gè)西南混作區(qū)馬鈴薯主栽品種進(jìn)行盆栽接種試驗(yàn)，以會(huì)薯16號(hào)作為感病對(duì)照。結(jié)果表明，云薯505、宣薯5號(hào)、會(huì)薯15號(hào)、會(huì)薯19號(hào)和云薯304共5個(gè)品種為高抗品種，抗性等級(jí)為9，馬鈴薯金線蟲(chóng)基本不能在這些品種上繁殖。麗薯6號(hào)和宣薯6號(hào)為中感品種，抗性等級(jí)為4。而抗性等級(jí)為1—3的7個(gè)品種均為高感品種（表3）。

感病對(duì)照會(huì)薯16號(hào)的最終單株孢囊數(shù)（Pf）為（223±36）個(gè)孢囊，平均繁殖系數(shù)（Pf/Pi）為17.15，表明馬鈴薯金線蟲(chóng)在該品種上大量繁殖。感病品種中，會(huì)-2、宣薯8號(hào)和麗薯15號(hào)的Pf大于對(duì)照，Pf/Pi分別為23.84、20.69和18.85。中感品種麗薯6號(hào)和宣薯6號(hào)Pf小于感病對(duì)照品種，Pf/Pi分別為3.31和4.08。而5個(gè)高抗品種的最終群體數(shù)量Pf僅為0—2個(gè)孢囊，Pf/Pi為0—0.15。

2.2 抗馬鈴薯金線蟲(chóng)H1的分子標(biāo)記檢測(cè)

采用相關(guān)分子標(biāo)記57R和TG689鑒定室內(nèi)接種試驗(yàn)的15個(gè)馬鈴薯主栽品種。以BCH擴(kuò)增的-胡蘿卜素羥化酶基因?yàn)閷?duì)照，所有品種均出現(xiàn)290 bp條帶，表明模板DNA無(wú)質(zhì)量問(wèn)題，可用于后續(xù)試驗(yàn)（圖1）。經(jīng)57R和TG689分子標(biāo)記擴(kuò)增共計(jì)5個(gè)品種出現(xiàn)450和141 bp大小條帶，即云薯505、宣薯5號(hào)、會(huì)薯15號(hào)、云薯304和宣薯6號(hào)（表3）。

表3 馬鈴薯品種對(duì)馬鈴薯金線蟲(chóng)抗性室內(nèi)鑒定評(píng)價(jià)及H1基因分子標(biāo)記檢測(cè)

a：BCH為陽(yáng)性對(duì)照BCH marker as positive controls；b：57R分子標(biāo)記，“+”與“-”表示經(jīng)檢測(cè)出現(xiàn)或未出現(xiàn)450 bp DNA片段57R marker, “+” and “-” indicate the presence and absence of 450 bp PCR products DNA fragments, respectively；c：TG689分子標(biāo)記，“+”與“-”表示經(jīng)檢測(cè)出現(xiàn)或未出現(xiàn)141 bp DNA片段TG689 marker, “+” and “-” indicate presence and absence of 141 bp PCR products DNA fragments, respectively

兩個(gè)基因鑒定分子標(biāo)記結(jié)果一致，西南混作區(qū)15個(gè)馬鈴薯主栽品種中5個(gè)馬鈴薯品種，即云薯505、宣薯5號(hào)、會(huì)薯15號(hào)、云薯304和宣薯6號(hào)含抗馬鈴薯金線蟲(chóng)Ro1和Ro4型的。

2.3 抗/感馬鈴薯品種田間抗性鑒定

馬鈴薯金線蟲(chóng)田間平均繁殖系數(shù)（Pf/Pi）＜1.00的品種共有7個(gè)，表現(xiàn)為抗病。5個(gè)抗性等級(jí)為9的高抗品種（云薯505、宣薯5號(hào)、會(huì)薯15號(hào)、會(huì)薯19號(hào)、云薯304）馬鈴薯金線蟲(chóng)Pf/Pi值最低為0.04—0.14；抗性等級(jí)為4的中感品種麗薯6號(hào)和宣薯6號(hào)的Pf/Pi值為0.43—0.65；8個(gè)高感品種Pf/Pi在1.18—3.24（圖2-c—f）。所有高感品種的Pf/Pi值在2021年（1.18—2.75）和2022年（1.76—3.24）均＞1.00，且均大于抗病品種，表明感病品種種植后田間線蟲(chóng)種群數(shù)量增加。相比之下，高抗品種的Pf/Pi值在2021年（0.04—0.12）和2022年（0.05—0.14）均＜1.00，表明這些抗病品種種植后馬鈴薯金線蟲(chóng)田間種群密度有所降低（表4）。

1：云薯505 Yunshu 505；2：宣薯5號(hào)Xuanshu 5；3：會(huì)薯15號(hào)Huishu 15；4：會(huì)薯19號(hào)Huishu 19；5：云薯304 Yunshu 304；6：麗薯6號(hào)Lishu 6；7：宣薯6號(hào)Xuanshu 6；8：合作88 Cooperation 88；9：會(huì)-2 Hui-2；10：宣薯8號(hào)Xuanshu 8；11：宣薯4號(hào)Xuanshu 4；12：宣薯2號(hào)Xuanshu 2；13：麗薯15號(hào)Lishu 15；14：麗薯7號(hào)Lishu 7；15：會(huì)薯16號(hào)Huishu 16

a、b：馬鈴薯品種田間抗性鑒定Identification of resistance of potato cultivars in the field，a：高抗品種云薯505 the highly resistant cultivar Yunshu 505；b：高感品種宣薯2號(hào)The highly susceptible cultivar Xuanshu 2；c：會(huì)薯16號(hào)受馬鈴薯金線蟲(chóng)侵染的地上部癥狀A(yù)bove-ground symptoms of Huishu 16 infested with G. rostochiensis；d—f：會(huì)薯16號(hào)根表的馬鈴薯金線蟲(chóng)雌蟲(chóng)、孢囊及2齡幼蟲(chóng)Females, cysts, and 2nd stage juveniles of G. rostochiensis on the roots of Huishu 16

表4 15份馬鈴薯主栽品種的田間抗性

2.4 抗/感馬鈴薯田間農(nóng)藝性狀比較

整體上，5個(gè)高抗品種株高的均值在兩年間均顯著高于8個(gè)高感品種的株高均值，且高抗品種產(chǎn)量的均值在兩年間也均顯著高于8個(gè)高感品種的產(chǎn)量均值（＜0.05）。宣薯5號(hào)、會(huì)薯15號(hào)和會(huì)薯19號(hào)產(chǎn)量相對(duì)較高，顯著高于其他馬鈴薯品種；會(huì)-2產(chǎn)量最低，顯著低于抗病品種，且低于或顯著低于其他感病品種。會(huì)薯16號(hào)、麗薯7號(hào)、宣薯8號(hào)和麗薯15號(hào)產(chǎn)量相對(duì)較低，與產(chǎn)量最低的會(huì)-2無(wú)顯著差異，但顯著低于其他馬鈴薯品種。此外，合作88和宣薯2號(hào)與云薯304和麗薯6號(hào)產(chǎn)量無(wú)顯著差異（表5）。

3 討論

3.1 馬鈴薯對(duì)馬鈴薯金線蟲(chóng)的抗性評(píng)價(jià)

馬鈴薯西南混作區(qū)自2013年產(chǎn)量超越北方一作區(qū)后，成為我國(guó)最主要的馬鈴薯生產(chǎn)區(qū)域[34]。而馬鈴薯金線蟲(chóng)在該區(qū)的發(fā)生危害嚴(yán)重限制了我國(guó)馬鈴薯的高產(chǎn)穩(wěn)產(chǎn)和貿(mào)易交流。目前，種植抗病品種仍是防控和遏制該線蟲(chóng)進(jìn)一步發(fā)展擴(kuò)散最為經(jīng)濟(jì)有效的方法。因此，本研究采用室內(nèi)接種鑒定對(duì)西南混作區(qū)馬鈴薯主栽品種進(jìn)行了抗性等級(jí)評(píng)價(jià)，并將分子標(biāo)記鑒定作為輔助手段，高效、可靠地篩選出5份高抗馬鈴薯金線蟲(chóng)品種?？剐缘燃?jí)為9的高抗品種上馬鈴薯金線蟲(chóng)田間繁殖系數(shù)極低，均＜0.15，表明馬鈴薯金線蟲(chóng)基本不能侵染或者不能在這些品種上完成生活史。而對(duì)于抗性等級(jí)≤3的高感品種，馬鈴薯金線蟲(chóng)田間繁殖系數(shù)則均＞1，甚至在品種宣薯2號(hào)和宣薯8號(hào)上經(jīng)一個(gè)生長(zhǎng)季節(jié)后馬鈴薯金線蟲(chóng)種群增長(zhǎng)2—3倍，表明連續(xù)種植感病品種可使田間馬鈴薯金線蟲(chóng)種群密度不斷擴(kuò)大，蟲(chóng)源基數(shù)逐年上升，病害發(fā)展逐年加重。馬鈴薯高抗品種的室內(nèi)盆栽接種和田間抗性鑒定的繁殖系數(shù)結(jié)果高度一致。高感品種的繁殖系數(shù)在盆栽和田間鑒定中均＞1，但盆栽中的繁殖系數(shù)高于田間；兩個(gè)中感品種在盆栽接種中Pf/Pi＞1而田間鑒定中Pf/Pi＜1，但其抗性水平在兩個(gè)試驗(yàn)中均介于高感和高抗品種之間，繁殖系數(shù)不一致的情況可能與不同的鑒定環(huán)境有關(guān)，如田間的線蟲(chóng)初始群體數(shù)量差異可能會(huì)影響繁殖率。

由于對(duì)馬鈴薯金線蟲(chóng)Ro1型展現(xiàn)出優(yōu)異抗性，在馬鈴薯抗病育種中得到廣泛應(yīng)用[17,35]。含有的抗病品種可能同感病品種一樣會(huì)誘導(dǎo)馬鈴薯金線蟲(chóng)的孵化。然而在線蟲(chóng)侵入根系后，抗病寄主中線蟲(chóng)的取食位點(diǎn)周?chē)募?xì)胞會(huì)觸發(fā)過(guò)敏反應(yīng)，使得合胞體（孢囊線蟲(chóng)誘導(dǎo)寄主根部形成的取食細(xì)胞）在誘導(dǎo)形成的一周內(nèi)發(fā)生退化，最終阻斷馬鈴薯金線蟲(chóng)2齡幼蟲(chóng)的營(yíng)養(yǎng)攝取而達(dá)到抗病且減少田間線蟲(chóng)群體基數(shù)的目的[36]。本研究表明抗馬鈴薯金線蟲(chóng)Ro1致病型的對(duì)我國(guó)馬鈴薯金線蟲(chóng)群體具有高水平抗性。利用室內(nèi)抗性鑒定和分子標(biāo)記能夠有效對(duì)馬鈴薯抗性進(jìn)行鑒別。室內(nèi)鑒定得到的5個(gè)高抗品種中均含抗性。中感品種宣薯6號(hào)雖然檢測(cè)含，但是室內(nèi)鑒定和田間試驗(yàn)均表明其抗性水平不及高抗品種。Biryukova等[37]和Faggian等[38]認(rèn)為馬鈴薯對(duì)金線蟲(chóng)抗性符合數(shù)量性狀遺傳特征，由少數(shù)主效基因和多個(gè)微效基因共同控制，品種在培育過(guò)程中微效基因控制的抗性在基因傳遞時(shí)可能發(fā)生重組，這可能是宣薯6號(hào)抗性水平發(fā)生改變的原因。此外，除外，抗金線蟲(chóng)Ro1致病型還有、和等抗性基因[18,33,39-40]，高抗品種會(huì)薯19號(hào)未檢測(cè)含有，表明其可能存在其他新型抗性基因發(fā)揮抗性作用。此次試驗(yàn)金線蟲(chóng)群體均來(lái)自云南省昭通市昭陽(yáng)區(qū)西魁坪子種薯基地，還需要進(jìn)一步根據(jù)馬鈴薯金線蟲(chóng)的發(fā)生分布，有針對(duì)性地?cái)U(kuò)大對(duì)當(dāng)?shù)刂髟择R鈴薯品種抗性程度的鑒定和評(píng)價(jià)。

表5 15份馬鈴薯主栽品種的農(nóng)藝性狀

同列數(shù)據(jù)后不同小寫(xiě)字母表示處理間差異顯著（＜0.05）

Values followed by different lowercase letters in the same column indicate significantly different between the cultivars at the 0.05 level

3.2 馬鈴薯抗/感品種對(duì)金線蟲(chóng)群體的影響

金線蟲(chóng)在馬鈴薯上的繁殖率很大程度上取決于初始種群密度[41-42]。當(dāng)金線蟲(chóng)群體密度＜1粒卵/g土?xí)r，群體便可增殖高達(dá)70倍，而當(dāng)線蟲(chóng)群體＞100粒卵/g土?xí)r，由于根系損傷和2齡幼蟲(chóng)之間的競(jìng)爭(zhēng)增加，增殖率反而受到抑制[23,41,43]。本試驗(yàn)同樣發(fā)現(xiàn)馬鈴薯金線蟲(chóng)在合作88和宣薯8號(hào)等感病品種上的繁殖系數(shù)在相對(duì)較低的初始群體密度下的增殖和繁殖系數(shù)相對(duì)較高。

國(guó)外研究認(rèn)為金線蟲(chóng)的經(jīng)濟(jì)閾值為20粒卵/g土，而該數(shù)值會(huì)受到土壤類型、肥力及產(chǎn)量水平等因素影響[43]。在英國(guó)，金線蟲(chóng)的經(jīng)濟(jì)閾值約為15粒卵/g土，若不進(jìn)行有效防控，最高可造成80%的產(chǎn)量損失[44]。在新西蘭，金線蟲(chóng)群體密度達(dá)到5—10粒卵/g土?xí)r田間才表現(xiàn)明顯癥狀，當(dāng)達(dá)到15—20粒卵/g土，對(duì)感病品種造成嚴(yán)重危害，最高可造成85%—90%的產(chǎn)量損失[45]。在云南和四川馬鈴薯金線蟲(chóng)群體種群密度為47—69粒卵/g土[6]，除與品種特性相關(guān)的重要農(nóng)藝性狀如株高等表征非常明顯外，在馬鈴薯金線蟲(chóng)危害嚴(yán)重田塊，感病品種產(chǎn)量大多極顯著低于高抗品種產(chǎn)量。

研究表明，種植含抗性基因的馬鈴薯品種能夠使金線蟲(chóng)Ro1致病型種群密度減少80%—90%[40,46-47]。雖然目前尚不明確我國(guó)馬鈴薯金線蟲(chóng)的致病型類別，但本研究表明含的抗性馬鈴薯品種能夠降低我國(guó)馬鈴薯金線蟲(chóng)田間群體密度。英國(guó)、新西蘭、美國(guó)、加拿大和塞爾維亞開(kāi)展的長(zhǎng)期試驗(yàn)的結(jié)果表明2—3年輪作或連作種植抗性基因馬鈴薯品種能夠?qū)⒔鹁€蟲(chóng)種群降至極低甚至難以檢測(cè)的水平[45-46,48]。但長(zhǎng)期單一大面積種植含抗性基因品種也會(huì)對(duì)田間馬鈴薯金線蟲(chóng)群體形成較高的選擇壓力，使得金線蟲(chóng)群體向其他致病型發(fā)展，導(dǎo)致抗性品種的抗性喪失[47]。面對(duì)馬鈴薯金線蟲(chóng)入侵我國(guó)的嚴(yán)峻形勢(shì)，未來(lái)應(yīng)將抗馬鈴薯金線蟲(chóng)納入馬鈴薯抗性育種目標(biāo)，合理布局抗病品種并結(jié)合輪作等措施對(duì)金線蟲(chóng)開(kāi)展可持續(xù)綜合防控。

4 結(jié)論

結(jié)合室內(nèi)盆栽接種和分子標(biāo)記鑒定了我國(guó)馬鈴薯西南混作區(qū)7個(gè)主栽抗病品種并結(jié)合田間抗/感病品種對(duì)馬鈴薯金線蟲(chóng)群體的影響及其農(nóng)藝性狀表現(xiàn)，篩選出宣薯5號(hào)、會(huì)薯15號(hào)和會(huì)薯19號(hào)為高產(chǎn)、抗病的良好品種?？柜R鈴薯金線蟲(chóng)Ro1致病型的對(duì)我國(guó)馬鈴薯金線蟲(chóng)群體具有高水平抗性且能夠減少田間馬鈴薯金線蟲(chóng)群體密度。而受馬鈴薯金線蟲(chóng)侵染田間種植的感病品種產(chǎn)量大多顯著低于高抗品種，且感病品種種植后會(huì)導(dǎo)致線蟲(chóng)種群基數(shù)增大病害加重。研究結(jié)果可為馬鈴薯金線蟲(chóng)抗病育種綜合評(píng)價(jià)提供參考，并為指導(dǎo)馬鈴薯金線蟲(chóng)綜合防控、抗病品種合理布局和品種示范推廣提供依據(jù)。

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Identification and evaluation of major potato cultivars resistance toand detection of theirresistance gene marker

HUANG LiQiang1, JIANG Ru1, ZHU BoZhi1, PENG Huan1, XU Chong2, SONG JiaXiong2, CHEN Min2, LI YongQing2, HUANG WenKun1, PENG DeLiang1

1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193;2Plant Protection and Quarantine Station of Zhaotong City, Yunnan province, Zhaotong 657000, Yunnan

【Objective】Potato cyst nematodes (PCN), includingand, are damaging soilborne quarantine parasitic pests on potatoes. Thehas now distributed in seven counties of Yunnan, Guizhou, and Sichuan provinces in China, and several seed potato bases are threatened by the spread of. This study aimed to identify the resistance level of major potato cultivars to, clarify the presence of a knownresistance gene, and to provide a reference for screening and promoting resistant cultivars for controlling.【Method】For bioassay, 15 major potato cultivars from the southwest mixed-cropping zone were inoculated withpopulations in an isolation greenhouse. The number of cysts per plant was counted and relative susceptibility was calculated, and finally the resistance of the cultivars was ranked according to a standard scoring notation. Forresistance gene identification, two molecular markers 57R and TG689, linked to thefor resistance toRo1 pathotype, were used to identify 15 cultivars of the bioassay, with the BCH marker for amplification of the beta carotene hydroxylase gene as a control. For the field trial, a comparison of resistant and susceptible potato cultivars was conducted in a naturally infested field in 2020 and 2021 in Zhaotong, Yunnan Province. To estimate the initial (Pi) and final (Pf) populations of, soil sample was taken before planting and after harvested, and the reproduction factor (Pf/Pi) was calculated. The height of above-ground part of the plant at budding and initial flowering stages and the total yields at harvesting stage were measured.【Result】Among the 15 major potato cultivars, five cultivars (Yunshu 505, Xuanshu 5, Huishu 15, Huishu 19, and Yunshu 304) are highly resistant. It’s hard forto reproduce on these cultivars. Two cultivars (Lishu 6 and Xuanshu 6) are moderately susceptible. Eight cultivars are highly susceptible, especially the Pf/Pi of Hui-2, Lishu 15 and Xuanshu 8 was higher than that of the susceptible control Huishu 16 (Pf/Pi=17.15). Additionally, the results of the twogene-linked molecular markers were generally consistent, five cultivars (Yunshu 505, Xuanshu 5, Huishu 15, Yunshu 304, and Xuanshu 6) contain the.reproduction factor differed significantly on resistant and susceptible cultivars in the field. The average Pf/Pi values of highly resistant cultivars (with resistance score 9) were ＜1 in 2021 (0.04-0.12) and 2022 (0.05-0.14), indicating that nematode population densities decreased after planting of highly resistant cultivars. On the contrary, the average Pf/Pi values of the highly susceptible cultivars were ＞1.00 in 2021 (1.18-2.75) and 2022 (1.76-3.24), indicating an increase in nematode populations in the field after planting of highly susceptible cultivars. There were significant differences between cultivars in plant height and yield (＜0.05). The mean height of the five highly resistant cultivars was significantly higher than that of the eight highly susceptiblecultivars in the two years. The cultivars Xuanshu 5, Huishu 15 and Huishu 19 had the highest yields of 51.67-56.48 and 33.28-40.57 t·hm-2in two years, while Hui-2 had the lowest yields.【Conclusion】The resistant cultivars in the southwest mixed-cropping zone show excellent resistance to, mainly carrying the, and can reducepopulation densities in the field. High yields are produced by the resistant cultivars Xuanshu 5, Huishu 15, and Huishu 19, while Yunshu 304 is utilized as a processing potato for zinc-rich crisps. More focused identification of resistance levels of local cultivars based onincidence is urgently required.

; potato resistant cultivar;resistant gene; molecular marker detection

10.3864/j.issn.0578-1752.2024.08.007

2023-11-27；

2024-01-04

國(guó)家重點(diǎn)研發(fā)計(jì)劃（2023YFD1400400）、國(guó)家自然科學(xué)基金（32072398）、云南省彭德良專家工作站（2023-126）、昭通市科學(xué)技術(shù)局彭德良專家工作站（2021 ZTYX03）、政府購(gòu)買(mǎi)服務(wù)項(xiàng)目（15190025）、中國(guó)農(nóng)業(yè)科學(xué)院科技創(chuàng)新工程（ASTIP-02-IPP-15）

黃立強(qiáng)，E-mail：hlq292573628@163.com。江如，E-mail：jiangruby@126.com。黃立強(qiáng)和江如為同等貢獻(xiàn)作者。通信作者彭德良，E-mail：pengdeliang@caas.cn

（責(zé)任編輯 岳梅）