
中圖分類號:S662.1 文獻標志碼:A 文章編號:1009-9980(2025)08-1811-09
Abstract: 【Objective】 Prunus persica (L.) Batsch,a member of the Rosaceae family,is native to China and has a cultivation and evolutionary history spanning thousands of years.In recent years,peach shoot blight caused by Diaporthe amygdali has been one of the most prevalent fungal diseases in major peachproducing regions of southern China. This study aimed to screen out highly effective chemical fungicides for controlling peach shoot blight, and investigated the relationship between volatile compounds in the phloem of peach shoots and resistance to the disease, providing a scientific basis for integrated disease management strategies and resistance breeding programs.【Methods】The inhibitory effects of 15fungicidesagainst D .amygdali were evaluated in vitro using the mycelial growth rate method. The active ingredient information wasas follows: difenoconazole,pyraclostrobin,propiconazole,boscalid, carbendazim,hymexazol,flusilazole,fludioxonil,hexaconazole,prochloraz,azoxystrobin,trifloxycide.The effective medium concentration( EC50) )valueswere calculated based on the correlation between fungicide concentration and mycelial growth inhibition rate by DPS in this study. At the same time,healthy and disease-free one-year-old shoot phloem tissues were collcted from 22 peach varieties,which exhibited varying levels of resistance to D . amygdali. After rapid freezing within the liquid nitrogen, the samples were transported to the laboratory for volatile compound analysis. Volatile compounds were extracted and analyzed using Headspace Solid-Phase Microextraction coupled with Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS). The correlation between the relative content of each identified volatile compound and the resistance level of the corresponding variety was examined.In addition,two typical peach cultivars (Fenghua Pantao, susceptible; and Hakuho, highly susceptible) were selected for inoculation with D . amygdali. Phloem tissue around necrotic spots was collected 7 days after inoculation, flash-frozen, and used for volatile compound analysis. Key volatile compounds significantly correlated with resistance (identified in the correlation analysis) were quantified in these tissues to observe pathogen-induced changes.【Results】 The toxicity test of fungicides showed that prochloraz had the strongest inhibitory effect, with an EC50 value of 0.0042mg?L-1 . Difenoconazole, pyraclostrobin, propiconazole, fludioxonil, flusilazole, hexaconazole,and tebuconazole also showed strong activity. Boscalid showed the weakest inhibitory effect, with an EC50 value of 49.0252mg?L-1 .Analysis of volatile compounds content showed that volatile compounds were identified in 22 peach samples. A total of 121 volatile compounds were identified,falling into 12 distinctcategories: 30 terpenoids,22 esters,17alcohols,16ketones,13aldehydes,5alkanes,4 fattacids,3phenolics,3furans,2acids,1lactone,and 5 other compounds.Among the samples,Richuan Baifeng and Reddomun had the highest number of volatile compounds, with 85and 80,respectively. In contrast, the samples Baihua and Zhouxingshantao had the fewest, with 52 and 51 compounds,respectively.To identify volatile metabolites associated with resistance to D . amygdali, we conducted a correlation analysis between the volatile substance content and germplasm resistance to D . amygdali across 22 samples. Sixteen compounds were significantly and negatively correlated with resistance,and six were significantly and positively correlated.Negatively correlated compounds included one acid,one aldehyde,two alkanes,two esters,one fatty acid,four ketones,one phenolic compound,and four terpenoids. The ester compound icosyl oleate had the strongest negative correlation, with a coefficient of -0.676 5 四 (Plt;0.01 ).Positively correlated compounds included one alcohol, two aldehydes, one ester, one ketone, and one terpenoid. Among these,the aldehyde 2-heptenal had the highest positive correlation,with a coefficient of 0.570 4 (Plt; 0.01).The distribution of two volatile compounds (a: n-Hexadecane; b: 1-Nonanol) with different resistance level to D .amygdali of 22 peach varieties was revealed. To further validate the relationship between volatile compounds and shoot blight resistance, we selected two peach cultivars with different resistance levels and inoculated them with D .amygdali.After 7 days,lesion diameters reached 52.62mm in Fenghua Pantao and 84.50mm in Hakuho. Volatile compounds in the phloem tissue around the lesions were analyzed in both samples. After inoculation, the content of the volatile compound 2-heptenal, positively corrlated with resistance,rapidly increased in the susceptible germplasm Fenghua Pantao,in comparison to the highly susceptible germplasm Hakuho,suggesting that higher 2-heptenal levels may enhance resistance.Benzoic acid, negatively correlated with resistance, was induced in both germplasms,but the increase was smallr in susceptible peaches,suggesting that lower accumulation may reduce resistance.【Conclusion】Among the 15 fungicides,prochloraz exhibited the strongest inhibitory effect, with an effective medium concentration value of 0.0042mg?L-1 .Correlation analysis identified 16 compounds that were significantly and negatively correlated with resistance,with the ester icosyl oleate showing the strongest correlation (r=-0.6765 ).Six compoundswere significantly and positively correlated,with 2-heptenal showing the highest correlation (r=0.5704 ).Thisstudylayed an important theoretical foundation for achieving sustainable comprehensive control of peach shoot blight.
Key Words: Peach shoot blight; Diaporthe amygdali; Fungicide screening; Volatile compound
桃[Prunuspersica(L.)Batsch]為薔薇科桃屬植物,起源于中國,在中國有幾千年的栽培和進化歷史。中國是桃生產大國,但是桃樹在生長發育過程中易受多種病菌的侵染,如葡萄座腔菌(Botryos-phaeriadothidea)引起的桃流膠病,桃瘡痂病菌(Venturiacarpophila)引起的桃瘡痂病,核果鏈核盤菌(Monilinialaxa)引起的桃褐腐病等μ]。近年來,桃枝枯病已經成為中國南方各大桃產區最主要的真菌病害之一,發病桃樹產量損失 20%~50% ,嚴重時甚至絕收,給桃農帶來巨大的經濟損失2。該病最早被外國學者于1905年報道3,1934年該病在美國新澤西州首次出現,之后在美國東部沿海地區均有發生[4-5]。該病在中國的報道較晚,最早于1989年在云南地區發現。該病主要危害新梢,通常在新梢基部出現褐斑,后呈環狀或向上擴展,致使葉片枯黃、脫落和新枝枯死,嚴重影響桃樹生長和果實產量,6月下旬至7月是該病害發生的高峰期。對于桃枝枯病的病原菌,Delacroix等于1905年最早將其命名為桃殼梭孢(Fusicoccumamygdali)。Tuset等在1989年認為發生于西班牙的桃枝枯病病原菌 (F. amygdali)形態和擬莖點霉屬(Phomopsis)一致,因此將桃枝枯病病原菌改名為桃擬莖點霉(Phomop-sisamygdali),其有性態為間座殼屬(Diaporthe)。在美國南卡羅來納州,引起桃枝枯病的病原菌有葡萄座腔菌、桃擬莖點霉、白殼菌屬以及殼囊孢屬。在中國桃枝枯病優勢病原菌為桃擬莖點霉[610]。
目前,桃樹枝枯病的防治主要依靠化學防治。國內外研究者對此進行了一些研究,如Uddin等[]研究發現,春季和秋季同時噴施殺菌劑,百菌清和克菌丹防效最佳,防效分別為 46%~71% 和 46%~69% 其次是嘧菌酯和腈菌唑,防效分別為 41% 和 28% 44% 。紀兆林等[2研究發現多效唑對枝枯病也有一定的防效。范昆等3測定了丁香菌酯、辛菌胺單劑以及不同比例的混配制劑對桃枝枯病菌的毒力和聯合毒力,結果表明丁香菌酯與辛菌胺的復配對桃枝枯病菌有協同增效作用。鑒于防治桃枝枯病的藥劑種類繁多,需要更多的殺菌劑篩選試驗來為該病害的防控提供技術支撐。
同時,植物在生長發育過程中為了保護自身免受有害細菌、真菌和線蟲的侵害,植物在進化過程中形成了通過釋放揮發性物質(volatileorganiccom-pounds,VOCs)的保護機制[14]。VOCs具有直接抑菌或誘導抗性的潛力,是生物防治研究的新方向[15]。同時VOCs作為植物重要的防御體系組成部分與抗性密切相關。然而,對桃樹枝枯病抗性與枝條韌皮部VOCs的關聯尚不清楚。為篩選防治桃枝枯病的高效低毒化學藥劑,并鑒定可能參與抗病性形成的代謝產物,筆者在本研究中通過室內毒力測定試驗篩選藥劑;通過韌皮部VOCs與桃枝枯病抗性的關聯分析明確正負相關VOCs;并通過接種試驗進行初步驗證。預期結果可為桃枝枯病的化學防治提供高效藥劑選擇依據,同時為基于VOCs的生物防治新策略及抗病育種提供理論支撐。
1 材料和方法
1.1材料
供試植物:22份桃種質的抗性詳細信息見表1。
供試菌株:桃擬莖點霉(Diaportheamygdali)由中國農業科學院鄭州果樹研究所果樹病害防控課題提供。
供試藥劑15種,分別為 95% 苯醚甲環唑(Difeno-conazole)原藥(上海生農生化制品股份有限公司)、98% 吡唑醚菌酯(Pyraclostrobin)原藥(陜西諾正生物科技有限公司) 95% 丙環唑(Propiconazole)原藥(山東東泰農化有限公司) .96% 啶酰菌胺(Boscalid)原藥(浙江禾本科技股份有限公司) 98% 多菌靈(Carben-dazim)原藥(山東濰坊潤豐化工股份有限公司) 99% 噁霉靈(Hymexazol)原藥(青島星牌作物科學有限公司) 95% 氟硅唑(Flusilazole)原藥(山東濰坊潤豐化工股份有限公司) 95% 咯菌晴(Fludioxonil原藥(山東濰坊潤豐化工股份有限公司) 95% 己唑醇(Hexa-conazole)原藥(山東濰坊潤豐化工股份有限公司)、
表122份桃種質對桃擬莖點霉菌的抗性
Table122peachgermplasmswithdifferentresistanttoPhomopsisamygdali

97% 咪鮮胺(Prochloraz)原藥(山東華陽農藥化工集團有限公司) 98% 嘧菌酯(Azoxystrobin)原藥(江陰蘇利化學股份有限公司)、 97% 腭菌酯(Trifloxys-trobin)原藥(山東華陽農藥化工集團有限公司) 97% 戊唑醇(Tebuconazole)原藥(山東濰坊潤豐化工股份有限公司)、 96% 異菌脲(Lprodione)原藥(山東濰坊潤豐化工股份有限公司)、 95% 二氰蒽醌(Dithianon)原藥(江西禾益化工股份有限公司)。
1.2殺菌劑毒力測定
15種殺菌劑對桃枝枯病菌的抑制效果測定。選擇15種殺菌劑(苯醚甲環唑、吡唑醚菌酯、丙環唑、啶酰菌胺、多菌靈、噁霉靈、氟硅唑、咯菌腈、己唑醇、咪鮮胺、嘧菌酯、肟菌酯、戊唑醇、異菌脲、二氰蒽醌)進行室內毒力測定,步驟為:稱取各種藥劑,用丙酮分別將原藥溶解,將溶解后的原藥分別用0.1% 吐溫-80水溶液配成 1% 的母液備用。用配好的母液,在預試驗的基礎上根據需要配置系列質量濃度。噁霉靈的系列終質量濃度為0.25、0.5、1、2、4、8,16mg?L-1 ,其他藥劑的質量系列終濃度為0.01、0.033、0.1、0.33、1、3和 9mg?L-1 。在無菌操作條件下,配置各藥劑的含藥PDA平板。每個處理3次重復,設置不含藥劑的處理作為空白對照。將培養好的桃擬莖點霉菌株接種到各平板的中央,放到 26°C 培養箱中培養。待空白對照長滿培養皿時,用數顯游標卡尺十字交叉測量各處理的菌落擴展直徑。
按公式(1)計算各濃度對病原菌菌絲生長的抑制率。

式中: :I 為菌絲生長抑制率; D0 為空白對照菌落直徑;
為藥劑處理菌落直徑。
1.3桃枝條韌皮部樣品采集
1.3.122份不接種病原菌韌皮部樣品的采集在王麗等前期的研究中,通過對桃枝枯病1年生枝條接種桃擬莖點霉后第7天的病斑長度進行調查,可將37份桃種質分為4類,其中高抗、抗、感和高感種質分別有5、11、15和6份。選擇其中的22份種質,收集其一年生新梢沒有接種病原菌部位的韌皮部組織,液氮速凍后帶至實驗室,用于揮發性物質含量的測定。不同種質來自同一個果園,栽培管理條件一致,樹體大小一致。種質抗性的詳細信息見表1。
1.3.22份接種病原菌韌皮部樣品的采集同時選擇其中的2個桃品種(奉化蟠桃和白鳳),用于接種桃枝枯病菌,接種方法參照王麗等[的報道。每個品種每次處理接種10個點,3次重復,以空白PDA培養基接種作為對照。接種后每天觀察發病情況,在接種7d后,用游標卡尺測量病斑直徑。采集處理后的枝條壞死斑周圍的韌皮部組織,用液氮速凍后,用于測定揮發性物質含量。
1.4枝條韌皮部揮發性物質含量的測定
稱取 5g 桃枝條韌皮部材料,液氮研磨后,轉移至樣品瓶中,加入 3mL20% CaCl溶液、 ,3mL 200mmol?L-1 EDTA溶液, 30μL 內標,渦旋混勻。40°C 平衡 30min 后,用 65μm Carboxen/聚二甲基硅氧烷(PDMS)固相微萃取頭萃取 30min 后進樣。
所用的GC-MS聯用儀為美國Agilent公司生產的7890-5975C型。MS離子源溫度 230°C ,離子化方式為電子轟擊電離,電子能量 70eV ,四級桿溫度為 150°C ,傳輸線溫度為 250°C ;進樣口:加熱器250°C ,壓力 48.9545kpa ,隔墊吹掃流量 3mL?min-1 ,進樣模式為不分流;色譜柱: 30mDB-WAX (安捷倫),流量 1mL?min-1 ,壓力 48.9545kpa ,恒定流量,后運行1mL?min-1 ;柱箱:柱箱溫度開啟 40°C ,初始值40°C 保持 2min ;以 3°C?min-1 升至 100°C ,再以5°C?min-1 升至 245°C ;輔助加熱器:開啟, 250°C ;時間 3min ,掃描起點50,掃描終點550,閾值150,掃描速度 1562u?s-1 ,溶劑延遲 3min 。
1.5揮發性物質的定量分析
質譜圖集采集結束后,在GC-MS系統打開NIST14.L質譜庫,在網站主界面信息檢索欄查找相關試驗測試數據,以質譜圖中的數據作為對比,根據測試數據的匹配度對揮發性物質進行定性分析。
揮發性物質相對含量計算方法為:組分含量(μg?kg-1)= 各組分的峰面積/內標的峰面積 × 內標質量 (μg)×1000/ 樣品量 Π(g) ,內標為仲辛醇。
1.6 數據分析
采用Excel軟件對試驗數據進行統計分析,根據各藥劑濃度對數值及對應的防效概率值作回歸分析,計算各藥劑的回歸方程、相關系數和有效中濃度( EC50 值。
2 結果與分析
2.1不同藥劑對桃枝枯病菌的毒力測定
測定了15種藥劑對桃枝枯病菌的毒力,結果表明,15種藥劑對桃枝枯病菌均有抑制作用,但 EC50 值差別比較大,在 0.0042~49.0252mg?L-1 之間(表2)。其中咪鮮胺的抑制作用最強,其 EC50 值為 0.0042mg?L-1 苯醚甲環唑、吡唑醚菌酯、丙環唑、咯菌腈、氟硅唑、己唑醇和戊唑醇對桃枝枯病菌也有較好的抑制作用,EC50 值均小于 1mg?L-1 ;其次為多菌靈、噁霉靈、異菌脲和二氰蒽醌, EC50 值在 1~10mg?L-1 之間;再次為肟菌酯和嘧菌酯, EC50 值分別為 14.379 2mg?L-1 和25.3709mg?L-1";啶酰菌胺對桃枝枯病菌的抑制作用最差, EC50"值為 49.0252mg?L-1"。
表215種藥劑對桃枝枯病菌的毒力測定
Table2ThetoxicityoffifteenfungicidestoD.amygdali

部分藥劑的防治效果如圖1所示,咪鮮胺、氟硅唑、苯醚甲環唑為抑制效果較好的3個藥劑;菌酯、嘧菌酯和啶酰菌胺為抑制效果較差的3個藥劑。
2.222份不同抗性桃種質韌皮部的揮發性物質含量分析
利用頂空固相微萃取-氣質譜聯用技術對22份桃種質(表1)進行揮發性物質含量測定,供試樣品可以檢測出121種揮發性物質。這些物質可以分為12類:萜烯類30種,酯類22種,醇類17種,酮類16種,醛類13種,烷烴類5種,脂肪酸類4種,酚類3種,呋喃類3種,酸類2種,內酯類1種,其他類5種。從單個樣品的揮發性物質個數上看,日川白鳳和紅甘露的揮發性物質種類最多,分別為85種和80種;白花和帚形山桃揮發性物質種類最少,分別為52和51種。

供試22份桃種質的總揮發性物質含量 (w) 范圍為46449.55~581121.78μg?kg-1 ,平均為 1279726.80μg?kg-1 總揮發性物質含量較低的3份樣品為帚形山桃、白鳳和湖景蜜露,含量分別為66229.10、66116.43和 46449.55μg?kg-1 ;揮發性物質含量較高的3份樣品為雨花露、紅甘露和中油蟠7號,含量分別為420 383.33、425 214.70和 581121.78μg?kg-1 0
2.3桃擬莖點霉菌抗性與其揮發性物質組分的相關性分析
為鑒定與抗桃擬莖點霉菌相關的揮發性物質,對22份桃種質的揮發性物質含量與該種質對桃擬莖點霉菌的抗性進行了相關性分析。發現有16種揮發性物質與抗性呈顯著負相關,6種揮發性物質與抗性呈顯著正相關。在顯著負相關物質中,包括1種酸、1種醛、2種烷烴、2種酯、1種脂肪酸、4種酮、1種酚和4種萜烯類物質,負相關系數最高的為酯類物質油酸二十烷醇酯,達到了 -0.6765(Plt;0.01) 。顯著正相關的物質包括1種醇、2種醛、1種酯、1種酮和1種萜烯類物質,其中正相關系數最高的為醛類物質2-庚烯醛,達到了 0.5704(Plt;0.01) 。
選擇其中兩種物質正十六烷和1-壬醇,分析其含量與抗桃擬莖點霉菌活性的相關關系,如圖2所示。其中,正十六烷在高感種質中的含量顯著高于其他抗性種質;而高抗種質中的1-壬醇含量高于感病種質,但與抗病、高感種質的含量沒有差異。

2.4桃枝枯病菌誘導桃枝條韌皮部的揮發性物質含量變化分析
為再次驗證揮發性物質含量與枝枯病抗性之間的關系,選擇對擬莖點霉抗性不同的2個桃品種接種病原菌。接種7d后觀察發現,奉化蟠桃和白鳳的病斑直徑分別為 52.62mm 和 84.50mm (圖3-A)。
圖32個桃品種接種擬莖點霉菌后的病斑大小 (A) 以及與抗性正(B)和負(C)相關的物質含量變化 Fig.3Changes in lesion size (A)and their compound contents related to positive (B)and negative (C)resistance of two peachvarietiesinoculatedwithD.amygdali

同時,對上述2個樣品病斑周圍的韌皮部組織進行揮發性物質含量測定,發現在接種病原菌后,與抗性呈正相關的揮發性物質2-庚烯醛含量在感病品種奉化蟠桃中快速上升,而在高感品種白鳳中上升幅度較小(圖3-B),表明2-庚烯醛含量的上升可能增強了品種的抗性。而與抗性呈負相關的揮發性物質苯甲酸含量在2個品種中都受到誘導,但在感病品種中上升的幅度低于高感品種(圖3-C),暗示該物質含量的上升降低了品種的抗性。
3討論
桃是中國重要的鮮食水果樹種,但枝枯病的發生嚴重影響了中國桃產業的可持續發展。目前,化學防治是防治桃枝枯病的主要方法,但長期不合理地使用化學藥劑會導致防效不理想,而加大農藥使用量則會造成農藥超標、病原菌產生抗藥性等問題。筆者通過對15種藥劑進行毒力測試,證實咪鮮胺對桃枝枯病菌的抑菌活性較強( EC50 值為0.004 2mg?L-1) ,與紀兆林等的報道高度一致( EC50 值為 0.0047mg?L-1) ,這為其田間應用提供了強有力的支持;而多菌靈 EC50 值為 2.4136mg?L-1 ,高于紀兆林等的結果 ?EC50 值為 0.0276mg?L-1) 。這種差異可能與病原菌群體對多菌靈抗藥性發展有關,在防治中需關注抗性風險并輪換用藥。雖然肟菌酯、嘧菌酯和啶酰菌胺屬較新型的藥劑,但其對桃枝枯病菌的抑制效果不如咪鮮胺及三唑類藥劑,并非防治桃枝枯病的首選藥劑。但這些藥劑殺菌譜廣,在防治果園的其他病害中仍有價值,如葡萄白粉病、灰霉病和腐爛病等,尤其啶酰菌胺對灰葡萄孢菌(Botrytiscinerea)引起的灰霉病抑制效果最佳[]。
植物中的VOCs可以有效地抑制多種植物病原菌的生長和繁殖,或者直接誘導植物的防御系統來抵御病原菌的侵害[18]。對于VOCs的抑菌作用已有大量研究。如Arroyo等發現反式-2-己烯醛是對草莓炭疽病菌絲生長抑制效果最好的一種VOCs。Guo等2研究表明,經反式-2-己烯醛處理后,番茄果實灰霉病的發病率顯著降低。香百里香酚和香芹酚在 125mg?L-1 時可完全抑制灰霉病[]。然而VOCs與桃枝枯病抗性的相關性研究報道較少,筆者首次發現桃枝條韌皮部VOCs中16種揮發性物質含量與抗性呈顯著負相關,6種揮發性物質含量與抗性呈顯著正相關,并鑒定出負相關系數最高的為油酸二十烷醇酯,正相關系數最高的為2-庚烯醛。高含量的負相關物質可能是感病性的標志,其可能競爭性地消耗了用于合成抗病相關化合物的共同前體,也可能干擾防御信號或利于病原菌定殖。枝條在接種擬莖點霉菌后,苯甲酸(負相關物質)在高感品種白鳳中的積累量高于感病品種奉化蟠桃,驗證了其在干擾防御或利于病原菌侵染方面的作用。而正相關物質的高含量與抗病性緊密關聯(如壬醛、1-壬醇、乙酸乙酯和2-庚烯醛),其防御機制可能是具有直接的抑菌活性,而在前人研究中,2-庚烯醛和1-壬醇曾被報道與黃曲霉(Aspergillusflavus)的生長相關[22-23],而壬醛和乙酸乙酯則分別與抗白色念珠菌(Candidaalbicans)和大麗輪枝菌(Verticilliumdahli-ae)相關[2425]。此外,正相關物質也可能起到防御反應誘導子的作用,或作為揮發信號介導間接防御。枝條在接種擬莖點霉后,2-庚烯醛含量在奉化蟠桃中上升速度快于白鳳,驗證了其作為主動防御響應的重要組成部分的結論。
4結論
在15種藥劑中,咪鮮胺對桃枝枯病菌的抑制作用最強,可為篩選適宜的化學防控藥品提供依據。同時,16種揮發性物質含量與抗性呈顯著負相關,負相關系數最高的為酯類物質油酸二十烷醇酯;6種揮發性物質含量與抗性呈顯著正相關,正相關系數最高的為醛類物質2-庚烯醛。本研究為未來開發新型生物防治制劑以及基于揮發物譜輔助篩選或創制高抗枝枯病的抗性桃品種奠定了重要的理論基礎,為最終實現桃枝枯病的可持續綜合防治提供了新思路和新方法。
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