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Studies on biological characteristics of Nalanthamala psidii and seedling inoculation method of guava
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|摘要:||番石榴 (Psidium guajava L.) 為熱帶和亞熱帶的重要果樹之一。目前記載有超過177種病原菌可引起番石榴病害，其中萎凋性病害是番石榴栽培產業的重要限制因子。在台灣可引起番石榴萎凋性病害的病原有 Nalanthamala psidii 與根瘤線蟲，而 N. psidii 為田間主要病原，該病原同時也在印度、馬來西亞及南非被記錄可引起番石榴萎凋病。在台灣，由 N. psidii 所引起的番石榴萎凋病，初期病徵為葉片黃化、捲曲及落葉，隨著病勢發展會出現梢枯、萎凋及立枯等病徵，因此又稱為立枯病。番石榴立枯病從1970年代開始造成嚴重產量損失，原因為農民因修枝產生的傷口，導致該病原菌易從傷口入侵植物組織。受感染的植株會在數個月後出現萎凋病徵，目前台灣尚未有推薦藥劑用於防治番石榴立枯病，主要以清園與傷口保護的方式降低病害的嚴重度與傳播。由於立枯病菌的接種主要以一年生以上嫁接苗或實生苗作為接種材料，且接種後需數個月以上才能確認測試菌株之病原性，因此十分耗時導致研究腳步緩慢。此外，立枯病菌在人工培養基上，可以產生短橢圓形與長橢圓形兩種分生孢子，然在田間僅能於已感染的植株上觀察到短橢圓形分生孢子的產生，此兩型孢子之生態特性尚未明瞭。本研究目的為嘗試發展快速檢測立枯病菌之接種方法，並尋找可產生短橢圓形或長橢圓形分生孢子之培養條件，作為研究其特性之方法。本研究自彰化、台南及高雄等地，共蒐集12 株可引起立枯病之菌株，經形態學特性與 ITS 序列比對後，鑑定為番石榴立枯病菌 N. psidii。比較嫁接苗與實生苗接種方法實驗中，結果顯示一年生嫁接苗接種法造成植株從發病到死亡約需 4-6 個月；番石榴實生苗以去頂芽製造傷口接種法使植株發病最快， 7 天即可產生病徵，而無傷口之頂芽接種則需 2 週才可觀察到病徵。至於，莖基部與剪根部位接種法，不會使植株產生病徵。進一步將罹病植株進行組織分離，在有外部病徵處皆可分離到病原菌，然無外部病徵分離率亦有 59.1 % 與 75 % ，顯示田間只將植株發病部位砍除，並無法有效達到清園的效果。為了瞭解最適接種濃度，將孢子懸浮液濃度配製成 100-107 conidia/ml，接種於去頂芽番石榴實生苗，結果顯示濃度高於 105 conidia/ml 時才可產生病徵，當高於 106 conidia/ml 時，植株會出現萎凋死亡的現象，因此後續實驗均以106 conidia/ml 作為接種濃度。本研究以去頂芽實生苗接種法測試 12 株供試菌株之病原性，所有接種植株在 2 週後都可觀察到病徵的產生，於第 4 週發病程度介於 38.9- 72.2 % 間，指出去頂芽實生苗接種法可用於快速檢測病原菌的致病能力，且結果亦顯示田間可能存在不同毒力菌株，後續試驗挑選毒力最強菌株 TD101 作為接種源。
番石榴立枯病菌於人工培養環境下可產生兩型分生孢子，為研究兩型孢子生物特性，依前人添加花楸糖或鼠李糖於液態 Czapek’s 培養液中進行培養，顯示兩型分生孢子皆可在液態培養產生。進一步以固態培養法測試 10 種碳素源對立枯病菌生長與產孢之影響。結果指出纖維素、蔗糖及海藻糖對立枯病菌生長最為良好，甘露糖、鼠李糖及葡萄糖次之，乳糖、半乳糖與木糖較差，然都可產生兩型分生孢子。唯以添加花楸糖之 Czapek’s 培養基於無光照下只可產生長橢圓形分生孢子，但其菌落形態會受到影響。立枯病菌培養於培養基中添加番石榴粗萃取液或滅菌釜消毒後之葉片中，無法誘發產生單一型的孢子，然接種於新鮮番石榴離葉片，卻可產生單一短橢圓形分生孢子。以去頂芽番石榴實生苗接種法評估長橢圓形孢子、短橢圓形孢子及兩型孢子混合後的致病能力的強弱，結果顯示接種長橢圓形分生孢子的番石榴幼苗在接種第 3 週後，只產生葉片黃化的病徵，發病度在接種第 4 週後達到 14.2 ％。然接種兩型分生孢子或接種短橢圓形分生孢子的番石榴幼苗，發病度在接種第 4 週可達到 47.6 % 與 52.3 ％。此結果證實短橢圓形分生孢子為立枯病菌在田間進行感染與傳播的重要構造。為瞭解引起植株產生萎凋病徵之原因，本研究以組織切片觀察接種部位與無外部病徵的組織，指出皮層及維管束組織內出現大量菌絲，且可觀察到菌絲末端有膨大現象，但組織無壞疽的現象發生。進一步以掃描式電子顯微鏡觀察，得知維管束中有類似孢子的構造存在。|
Guava (Psidium guajava L.) is one of important fruit crops in tropical and subtropical areas. More than 177 plant pathogens had been reported in guava worldwide. Among these diseases, the wilting diseases of guava are the limitation factor in guava production. In Taiwan, the causing agents of wilt disease include Nalanthamala psidii and Meloidogyne incognita, and the N. psidii is a major pathogen and widely distributed over the India, Malaysia and South Africa. The initial symptom of guava plant caused by N. psidii showed leaf yellowing, curling and defoliation, and the infected guava plant showed dieback and wilting later, called “Li-Ku-Bin”, in Taiwan. The guava wilting had seriously impaired the yield of guava since the 1970s due to pruning and the N. psidii penetrating into tissue easily. The infected guava plants showed wilting symptom after several months. Presently, no fungicides are recommended to control guava wilting disease, and the sanitary and wound treatment are common strategies on controlling guava wilting disease and decreasing the disease severity and transmission of pathogens in the field in Taiwan. The one-year-old grafted/non-grafted guava plant had been used to examine the pathogenicity of N. psidii. However, the wilting symptom showed after inoculation. Thus, the inoculation of N. psidii on one-year-old guava plant is time consuming and interrupts the study on N. psidii. Moreover, the N. psidii could produce two types of conidia in artificial medium, including long-elliptical conidia (LC) and short-elliptical conidia (SC). The SC can be produced in nature condition; meanwhile, the LC can not be produced in the field. Thus, the roles of the two types of conidia of N. psidii in ecology are still unknown. The objectives of this study were to develop a rapid, efficient inoculation method for examining the pathogenicity of N. psidii and to select the optimum condition for producing two types of conidia of N. psidii. A total of twelve isolates of N. psidii were obtained from Changhua, Tainan and Kaohsiung, and these isolates were identified as N. psidii based on morphological characteristics and ITS sequences. Guava seedling inoculation methods were tested to assess the wilt disease response on one-year-old grafted guava and seedling from seed. The results showed that the one-year-old grafted guava plants were died within 4 to 6 months after inoculation. The other side, the apical bud with wound of guava seedling showed symptom 7 days after inoculation, and the apical bud without wound showed symptom 2 weeks after inoculation. However, the basal stem and cutting-root did not have any symptom after inoculation. The reisolation rates of the N. psidii from the tissue with or without symptom were 59.1% and 75%, respectively. Thus, removing the parts showed symptom can not take off the source of N. psidii completely in the field. Several conidia concentrations (range from 101-107 conidia/ml) were used for the pathogenicity test. Result revealed that the 105 conidia/ml is limitation on showing symptom in guava seedling, and the guava seedlings were die after inoculation at a concentration of 106 conidia/ml. In this study, twelve isolates of N. psidii were inoculated on apical bud with wound of guava seedling and the symptoms could be showed 2 weeks after inoculation. Moreover, the disease severities of guava seedling inoculated by 12 isolates were ranged from 38.9 % to 72.2 %. Consequently, the apical bud with wound of guava seedling can use as material to examine the pathogenicity of N. psidii from field, and the result demonstrates that the virulent variations exist in isolates of N. psidii. Nalanthamala psidii can produce two types of conidia on artificial conditions. For carrying out the biological characters of the two types of conidia, we followed the previous study for producing LC in Czapek’s liquid medium added with rhamnose and L-sorbose. However, the two types of conidia could be produced simultaneously. In this study, the Czapek’s liquid medium was changed into Czapek’s solid medium and added with 10 different carbon sources. Of ten carbon sources tested, 9 carbon sources could induce TD101 isolate to produce two types of conidia except for L-sorbose. L-sorbose could only induce LC on Czapek’s solid medium at dark condition. The extraction of guava leaf and the guava leaf sterilized by autoclave did not induce SC production of TD101 isolate, but the fresh leaves did. For comparing the pathogenicity of SC and LC, the apical bud with wound of guava seedlings were inoculated by two types of conidia alone or in combination. The results demonstrated that LC could cause symptom 3 weeks after inoculation and disease severity was 14.2 % 4 weeks after inoculation. However, the SC alone and combination of two conidia could cause symptom rapidly and disease severity was 47.6% and 52.3% 4 weeks after inoculation. Thus, SC is major structure for infection and transmission in the field. The tissue section indicated that the TD101 isolate could produce mycelia and expansion hyphae in cortex and vascular bundle without symptom. Moreover, the SEM observation showed that conidia-like structure could be produced in vascular bundle.
|Appears in Collections:||植物病理學系|
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