云南省火龙果采后果腐病研究

果树学报2014，31(1)：111114 Journal of Fruit Science 云南省火龙果采后果腐病研究 郭力维 ，吴毅歆 ，何汉兴 ，毛自朝 ，何鹏博 ，何月秋1,2 ( 云南农业大学植物保护学院，昆明650201； 云南农业大学农学与生物技术学院，昆明650201； 纽约州立大学生物系，美国12561) 摘 要：【目的】探明火龙果(Hylocereus costaricensis)果腐病中的一种新的病原菌。【方法】以云南省元江水果市场上 发现的一种新病害为供试材料，应用分离培养、柯赫氏法则、形态学观察、寄主范围研究和ITS序列测定，对病原菌进 行鉴定。I结果】该病由霜霉目白秀科桃吉尔霉(Gilbertellapemicaria)引起，病原菌侵入火龙果2-3 d后最初呈现淡褐 色、水渍状小病斑；随即接种点病斑不断扩大，6 d后至使整个果实腐烂，病果表面长出大量浅白色菌丝体和淡褐色 孢子囊。该病原菌致病力受宿主种类和品种影响，接种3 d后的红肉火龙果发病程度较白肉火龙果重，且能引起带伤 口桃、梨果实腐烂。桃吉尔霉在30、黑暗条件下生长最好。【结论1-文首次报道我国火龙果采后果腐病由桃吉尔霉 引起以3O、黑暗为最适生长条件，宿主范围广，致病力强。 关键词：火龙果；桃吉尔霉；采后病害 中图分类号：$6679 文献标识码：A 文章编号：10099980(2014)01一叭11-04 A new fruit rot disease in Hyocereus costaricensis in Yunnan Province of China GUO Liwei ，WU Yixin ，HE Hanxing。，MAO Zichao ，HE Peng-bo ，HE Yue-qiu t (1Facuhy ofPlant Protection，Yunnan A cultural University，Kunming，Yunnan 650201 China；。Faculty Agronomy and Biotechnology， YunnanAgricultural University，Kunming，Yunnan650201 China；3Department ofBiology，State UniversityofNewYork，NewPaltz，NY 12561 USA) Abstract：Objective】The objective of the study is to identify a new fruit rot disease in dragon fruit(Hy locereus costaeicensis)during storage【Method】Dragon fruit infected by a new disease was used as ma terial and the pathogen was identified by cultural and morphological studies，pathogenicity tests and ITS sequence analysisResultThe resuhs showed the fruit rot disease was caused by Gilbertella persicaria Dragon fruit showed light brown water-soaked spots 2 to 3 d after inoculation of the pathogenThe lesion expanded to the whole fruit，which was covered with abundant sporangia and mycelia within 6 daysInoc- ulation studies showed that pathogenicity was depended on host species，and lesions were produced only on peach，pear and wounded tomato 3 days after inoculationRotting was more serious on fruits of H costariceusis with red skin and redpurple flesh than those of Hundatus with red skin and white flesh Fungal growth was optimal in 30and dark condition【ConclusionThis is the first report of Gpersicaria causing fruit rot of dragon fruit in ChinaThe pathogen can infect a wide range of fruit hosts especially in 30and dark conditionsTo minimize the disease，dragon fruit should be stored at low temperature and in uncovered containers Key words：Dragon fruit；Gilbertellapersicaria；Picked disease Hylocereus costaricensis Webb，Britton&Rose， commonly known as dragon fruit or pitaya，is a member of the family CactaceaeThe fruit has gained much in- terest not only because of its exotic features，attractive colors and great potential health benefits，but also be- cause of its high potential especially in areas where 收稿日期：20130616 接受日期：2013一l029 基金项目：国家国际科技合作项目2009DFA32360 作者简介：郭力维，女，在读硕士生，研究方向：植物病害生物防治。Email：g87112w12163COB 通信作者Authorfor correspondenceE-maih ynfh2007163corn 112 果 树 学 报 31卷 drought is a limiting factor for the production of other fruits” Dragon fruit was introduced to China in the 1990s and is now being widely cultivated in South China，in cluding provinces of Hainan，Guangdong，Guangxi， Fujian，Guizhou and YunnanIn the past，researchers focused primarily on its nutritional values with less at- tention paid on diseases in dragon fruitHowever，vari OUS diseases have been reported recently，such as stem blight(Diplodia sp，Ascochyta sp，Phoma sp)，soft rot(Erwinia sp)，canker(pathogen unknown)，an thracnose(Colletotrichum sp，Gloeosporium sp)and scab(a bacterium)，black spot(A Iternaria sp)，wilt (Fusarium oxysporium)，speck blight(Nectriella sp) ，stem rot( semitectum， oxysporium，F monil一 e)，basal rot (Pythium sp)and brown spot (Botryodiplodia sp) This paper reports a new pathogen causing fruit rot on dragon fruitThe objective was to isolate and identify the pathogen causing the disease found in Yuanjiang County of Yunnan Province，ChinaThis information will be helpful for the establishment of effective disease management Materials and Methods 11 Isolation of the pathogen The rotted fruit was collected from Yuanjiang County of Yunnan ProvinceNumerous sporangia were found on invasion sites(PlateD)With the help of a dissecting microscope at 200magnificationsingle sporangium was isolated with a sterile needle and transferred to a 9 cm diam Petri dish containing fresh potato sucrose agar(PSA)mediumAfter incubating at 25for 2 to 3 dsmall mycelial agar discs from the growing edges of the fungal colony were transferred aseptically onto new PSA plates 12 Characteristics of the pathogen 121 Identification The pathogen was identified by its morphological characteristics and nucleic rDNA se quence analysesFor its DNA extraction，about 25 mg frozen mycelia from a 4-day old culture on PSA were ground into fine powder in liquid nitrogenITS of fungal genome were amplified and sequenced as described by Tao et all51Homology analysis of the sequence was per- formed with the help of Blast(http：wwwncbinlmnih govblast)in NCBI 122 Fungal growth under different temperature and light conditions For examining effect of temperature， mycelia discs punched by a sterile cork-borer(70 mm diam)were transferred onto 9 cm diam PSA plates and incubated at 5，10，15，20，25，30，35 or40After the agar surface was fully covered by the fungus in one of the plates，the colony diameters of all plates were measured by the cross-cross methodThe examination of light effect on fungal growth involved the incubation of inoculated PSA plates at 25。(=in two separate incu bators，with one maintained in darkness，and another with 1 2 h alternating lightdark cyclesThree days lat er，the colony diameters were similarly measuredEach treatment was repeated 4 times 123 Pathogenicity This was tested on different fruits，including dragon fruit，summer squash，sweet potato，tomato，sponge gourd，cucumber，pumpkin， Chinese watermelon，peach and pearMycelia were picked from the margin of a flesh colony and transferred onto the surface of healthy，asymptomatic，unwounded or artificially wounded fruitsEach inoculated fruit was placed in a beaker with a sterilized wetted paper towel on the bottom，sealed with cellophane on the top and incubated at 25The experiment was repeated twice As for the contro1fruits were similarly inoculated each with a plain PSA agar disc Because different subspecies might vary in resis tance to the pathogen，two species of dragon fruits were tested by the same procedure as described above：H undatus HawBritton&Rose fruits with red skin and white flesh and Hcostaricensis fruits with red skin and redpurple fleshThree fruits were used per treatment and the experiment was repeated twice 2 Results and Analysis 21 Symp tom on Dragon fruit infected with the pathogen Both unwounded and artificially wounded dragon fruits developed lesion 2 to 3 d after inoculation and the symptoms were consistent with those found in the mar ketInitially，a small，light brown，water-soaked spots appeared on the inoculated sites and then the lesion ex 郭力维等：云南省火龙果采后果腐病研究(英文) 113 panded to the whole fruit，which was covered with abundant sporangia and mycelia within 6 daysThe dis ease was more serious on wounded than on unwounded fruits(PlateA to C)The same pathogen was reiso lated，fulfilling the Kochs postulates 22 Morphological characteristics of the pathogen The fungal colony was white，appressed and en- tirely covered a 9-cm diam PSA plate in less than 5 d at 25(PlateE)The sporangiophore was hyaline， light brown to grayish，non-septate，arising from the substrate hyphaeCalcium oxalate crystals were f0und covering the surface of the sporangiophores(Plate-F) and sporangia(PlateG)The sporangium was spheri cal，single and terminal on a sporangiophore which was 851(447 to 1431)Ixm in lengthThe sporangia were yellow-brown to brown when young and turned dark brown to black at maturity(Plate-H)As soon as a sporangium was transferred to a drop of water on a glass slide，its wall broke immediately releasing hundreds of sporangiospores(Plate-I) The sporangial wall then split into two halves along a longitudinal suture(Plate-J)With the abscission of the sporangial wall，a large pyriform columella was ex- posed on top of the sporangiophore(Plate-K)The sp0rangi0spores were mostly global to ellipsoidal，mea suring 51(37 to 78)p,m long and 63(41 to 92) m wide and nonseptateSome fine，hyaline appendages were found attached to the ends of the sporangiospores (PlateL)A1l measurements were based on at least 200 random samplesNo zygospores were found either on culture plates or infected fruits 23 Pathogenicity of the fungus Based on artificial inoculation，the fungus could also infect peach and pear fruits producing similar dis- ease symptoms(PlateM，N)Three days after inocula tion，the disease symptoms developed on wounded tomato but not on nonwounded tomato or artificially wounded fruits of sweet potato，pumpkin，summer squash，Chinese watermelon，sponge gourd and cu cumber 24 Cultural characteristics of the pathogen After 3 days of cultivation，the colony diameters at different temperature were recordedThe results indi- cated that the organism grew fastest at 30but could not grow at 5，10and 40 (Fig1)The fungus grew faster in dark than under a 12-h daily light dura- tionIn the dark incubator，the colony diameter almost was 90 cm 3 days later，while the colony in 12-h lightdark cycles just reached 78 cm(Plate-O，P) 0 宣 皇 00 80 60 40 20 00 50 100 150 200 250 300 350 400 Temperature Fig1 The effect of temperature on mycelium growth of the pathogen 25 Molecular characteristics of the Based on the ITS sequence pathogen (Accession Na JQ95 1 60 1)，the pathogen showed 98homology with Gilbertella persicariaIn terms of colony physiognomic characteristics，the pathogen was so similar to Rhizo pus nigricansCombining Benny search in 1991 about Gilbertellaeeae which gave details about this family with the molecular results，the pathogen was identified as Gilbertellapersicaria(Eddy)Hesseltine 26 Severity of the pathogen on different sub- species of dragon fruits Both species of dragon fruits developed disease af- ter inoculation，but the disease was more serious on fruit of Hcostaricensis than those of Hundatus (Plate-Q，R) 3 Discussion Based on the growth and morphological character istics，the fungus isolated from rotted dragon fruit in Yun nan Province was similar to Gilbertella persicaria(Ed- dy1 Hessehine as described by Benny嘲Its pathogenicity to dragon fruit by artificial inoculation followed by sue- cessful reisolation of the fungus and its identification by rDNA-nucleic analysis confirmed that dragon fruit rot was caused by Gpersicaria The fungus had been isolated from various sub strates like swine dung and soil in ChinaThe posthar vest decay of fruits was commonly associated with fun一 暑 】 声2 gnl_【j0 量_0 1l4 果 树 学 报 31卷 gal pathogens，such as Monilinia spand Rhizopus spp As a member of the order Mucorales，Gpersicaria was primarily known as a postharvest rotting pathogen in pear and peach as well as apricot，apple and tomato Although the sporangiospores of Rhizop us sppare larger than Gpersicaria，the symptoms of fruit decay caused by Rhizopus and Gilbertella are easily con fused，therefore accurate diagnosis is needed for effec tive plant disease management As far as we know，recent studies revealed that stem rot of pitaya was caused by Fusarium，Pythium， A cremonium and ophthora in Shanghai8After harvesting，dragon fruit are commonly infected by Bipolaris cactivora in Japan91 and Vietnam，Enter obacterloacoin Peninsula Malaysia1l1as well as Fusarium oxysporum and dimerum in Shanghai嘲 This is the first report of Gibbertella pericaria causing dragon fruit rotAlthough the fungus was reported to cause decay of tomato fruit ，the present study showed that it could cause fruit rot in tomato only when the fruit was woundedThe discrepancy may be attributable to the differences in the fungal isolates and tomato fruit used as well as the procedure of pathogenicity tests This pathogen grew faster at higher temperatures as reported by Ginting et a117，indicating that it adapts well to the tropical and subtropical areasThe present study also showed that the growth rate was higher in darkness than in lightBased on the inoculati0n resuhs it grew very fast on dragon fruit，especially on the fruit of HcostaricensisThese results suggest that dragon fruits should be kept at low temperature and in uncov ered containers during transportation from farm to the marketFurthermore，if possible，the fruit should not be transported among markets to prevent the disease from spreading to other places where no dragon fruit rot has yet been foundThe absence of zygospores in pre sent study suggests that the pathogen is probably het erothallic E 】and vigilant efforts must be maintained t0 prevent the import of the mating strains which could cross with the present strain to produce more virulent typesA brief report on the storage rot of dragon fruit caused by Gilbertella persicaria has been published re cently as a short note 3】 4 Conclusion The results suggest that Gpersicaria is able to in feet the dragon fruit especially in warm circumstances This findings would provide a foundation in under standing the storage conditions of dragon fruit(本文图 版见封3) References： 1】MIZRAHI Y，NERD A，SITRIT YNew Fruits for Arid ClimatesM In：Trends in New Crops and New Uses，ASHS PressUSA2002： 378-384 【2YUAN C L，ZHANG W F，AND YUAN H XPreliminary Report on investigation and control practices for pitaya diseases in Yuexi Pre fectureJSouth China Fruits，2004：33(2)：4950 【3】WANG D F，WEI Q，YANG R，SANG W J，FAN G Q，JIN Y L Pre