Evaluation of resistance to powdery mildew disease in cucurbits

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF AGRONOMY UNDERGRADUATE THESIS TITLE: EVALUATION OF RESISTANCE TO POWDERY MILDEW DISEASE IN CUCURBITS Student: Nguyen Thi Thu Class: K62KHCTT ID Student: 621817 Supervisor: PhD Ha Viet Cuong Department: Plant Pathology HA NOI, 2022 ACKNOWLEDGEMENTS I would like to express my great appreciation to my supervisor, Professor Dr Ha Viet Cuong for his supervision during my study The important advices, valuable comments, suggestions, and corrections from my supervisor were very helpful in order to finalize this dissertation I am appreciative of my class mates for sharing these wonderful years in the university with me I am thankful to Vietnam national University of Agriculture, faculty of Agronomy who in their unique ways, taught me more than plant diseases and pathogens Without the persistent encouragement that motivates me, I simply would not have accomplished this goal The love and gratitude I have for all of them cannot be put into words Much thankfulness is given to Field Crops Research Institute for providing seeds for my study I am grateful my parents for helping me find strength and teaching me the value of hard work, for their great sacrifice to provide me with an education I am especially thankful to my uncle, who always motivate me when I felt like giving up and never stopped believing me Nguyen Thi Thu i TABLE OF CONTENTS Acknowledgements i Table of contents ii List of tables v List of figures vi Abstract x PART INTRODUCTION 1.1 Introduction 1.2 Objectives and requirements of the study 1.2.1 Objectives 1.2.2 Requirements Part II LITERATURE Review 2.1 THE FAMILY CUCURBITACEAE .4 2.1.1 Use and economic importance .4 2.1.2 Taxonomic Classification 2.1.3 Cucurbit resistance to powdery mildew 10 2.2 POWDERY MILDEW FUNGI 11 2.2.1 Importance of powdery mildew 11 2.2.2 Research of powdery mildew in cucurbits in the world 13 2.2.3 Powdery mildew diagnosis and research 16 2.2.4 Research of powdery mildew in cucurbits in Vietnam .17 Part Iii MATERIALS AND METHODS 19 3.1 Content 19 3.2 Duration and location 19 3.3 MATERIALS 19 3.3.1 Polymerase chain reaction (PCR) material 19 3.3.2 Inoculation materials 20 3.4 methods 21 3.4.1 Plant genomic DNA extraction 21 ii 3.4.2 Fungal genomic DNA extraction .22 3.4.3 Amplification of ITS region from fungi by PCR 22 3.4.4 Sequencing of ITS region 22 3.4.5 Sequence analyses of ITS region 22 3.4.6 PCR analysis for CAPS markers 23 3.4.7 Analysis of PCR products by restriction enzyme 23 3.4.8 Resistance evaluation of cucurbit lines by inoculation .23 PART IV RESULTS AND DISCUSSION 26 4.1 IDENTIFICATION OF POWDERY MILDEW FUNGU ON CUCURBIT 26 4.1.1 Morphological characteristics .26 4.1.2 Molecular analysis 27 4.2 Evaluation of resistance to powdery mildew on pumpkin (Cucurbita pepo) 29 4.2.1 Evaluation of resistance to powdery mildew on pumpkin (Cucurbita pepo) in plastic pots 31 4.2.2 Evaluation of resistance to powdery mildew on pumpkin (Cucurbita pepo) in leaf disc 36 4.2.3 Identification of resistant gene to powdery mildew on pumpkin (Cucurbita pepo) by molecular maker 37 4.3 Evaluation of resistance to powdery mildew on Cucumis melon 44 4.3.1 Evaluation of resistance to powdery mildew on Cucumis melon in plastic pots 44 4.3.2 Evaluation of resistance to powdery mildew on C melon in leaf disc 49 4.3.3 Identification of resistant gene to powdery mildew on Cucumis melon by molecular maker 50 4.4 Evaluation of resistance to powdery mildew on Wax gourd (Benincasa hispida) 55 4.4.1 Evaluation of resistance to powdery mildew on Wax gourd in leaf discs 59 4.4.2 Identification of resistant gene to powdery mildew on Wax gourd by molecular maker 61 4.5 Evaluation of resistance to powdery mildew on Cucumber 66 iii 4.5.1 Evaluation of resistance to powdery mildew on Cucumber in plastic pots .66 4.5.2 Evaluation of resistance to powdery mildew on Cucumber in leaf discs 69 V CONCLUSION .71 5.1 CONCLUSION 71 5.1.1 Identification of powdery mildew fungus on cucurbits .71 5.1.2 Evaluation of pumpkin resistance .71 5.1.3 Evaluation of melon resistance 71 5.1.3 Evaluation of wax gourd resistance .71 5.1.4 Evaluation of cucumber resistance 72 5.2 RECOMMENDATION 72 REFERENCE .73 iv LIST OF TABLES Table 2.1 Major cucurbit producing countries and production estimates for 2009 (Source: FAOSTAT) Table 2.2 Cucurbit production in Vietnam for 2019 season Table 2.3 Currently recognized genera of Powdery Mildew .12 Table 3.1 The PCR primers used in the study 20 Table 4.1 BLAST search using the ITS sequences of the two powdery mildew samples (PT1 and PT2) 28 Table 4.2 Disease severity results of artificially inoculated pumpkin lines in plastic pots through seven times assessment The data were collected after 5days inoculation PM spores (N =10) 32 Table 4.3 Summary of results of detection of P xanthii antifungal gene on pumpkin selected by molecular markers (bp) 42 Table 4.4 Disease severity results of artificially inoculated melon lines in plastic pots through seven times assessment The data were collected after 5days inoculation PM spores 46 Table 4.5 Summary of results of detection of P xanthii antifungal gene on melon selected by molecular markers 53 Table 4.6 Disease severity results of artificially inoculated wax gourd lines in plastic pots through four times assessment The data were collected after 5days inoculation PM 56 Table 4.7 Disease severity result of artificially inoculated wax gourd lines in conditions of leaf disc The data were collected after days following inoculation 60 Table 4.8 Summary of results of detection of P xanthii antifungal gene on wax gourd selected by molecular markers 64 Table 4.9 Disease severity results of artificially inoculated cucumber lines in plastic pots through four times assessment The data were collected after 5days inoculation PM spores 67 Table 4.10 Disease severity result of artificially inoculated cucumber gourd lines in conditions of leaf disc The data were collected after days after inoculation 70 v LIST OF FIGURES Figure 2.1 Relationships of eight families in Cucurbitales based on molecular studies (Schaefer and Renner, 2011) Figure 2.2 Figure 2.3 Distribution of Cucurbitaceae Worldwide (Cucurbitaceae, 2015) Some kind of pumpkins belong to five major domesticated Cucurbiteae species C moschata and C argyrosperma (Brown et al., 2013); C ficifolia (Barrera-Redondo et al , 2020); C maxima (López-Anido, 2021); C pepo (Paris et al., 2015) Figure 3.1 Figure 4.1 Figure 4.2 Resistance evaluation of cucurbits by leaf disk inoculation 24 Symptoms and morphology of conidiophore, conidia and fibrosin body of powdery mildew fungus observed in pumpkin leaves at Gia Lam, Hanoi 27 Phylogenetic analysis based on ITS gene regions of powdery mildew fungi Two fungi samples collected on pumpkins in Gia Lam (PT1 and PT2) were marked The tree was constructed using MEGAX software based on genetic distances determined by the Neighbor-Joining method Only bootstrap values > 50% (1000 iterations) are shown The bar indicates genetic distance 29 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Artificial infection of P xanthii fungus on cucurbits plants after infection 30 Powdery mildew on some pumpkin plants after infection 31 Disease severity of artificially inoculated pumpkin lines in plastic pots through seven times assessment The data were collected after 5days inoculation PM spores The red arrows represent lines with potential resistance Blue arrow indicated lines that not carry resistant gene 36 Disease severity of artificially inoculated pumpkin lines in conditions of leaf disc The data were collected after days following inoculation Red arrows indicate potential lines carrying resistance genes 37 A) Position of primers of indicator S9_1539675 and position of enzyme MspI (=HpaII) on loci LOC111803649 (C pepo) and LOC111477906 (C maxima) B) Genotype related to Pm-0 vi resistance gene tested by indicator S9_1539675; R (resistant, homozygous dominant), S (infected, homozygous recessive) and H (resistant, heterozygous) (Holdsworth et al., 2016) 38 Figure 4.8 Detection of Pm-0 resistance gene by marker S9_1539675 (primers S9_1539 675-F and S9_1539 675-R) on selected pumpkin lines M1 is O'GeneRuler DNA Ladder Mix (Thermo Scientific) Red arrows represent incompletely cleaved PCR product Blue arrows indicate PCR products of unknown origin 39 Figure 4.9 Position indicator associates with the powdery mildew resistant QTL P xanthi on chromosome of TG10 (C moschata) The number on the scale bar is the nucleotide position (Park et al., 2020) 39 Figure 4.10 PCR products of five selected lines showing size difference Detection of genes for resistance to Pm-0 and QTL of powdery mildew resistance by marker MR2 (primer pairs MR2-F and MR2R) on selected pumpkin lines PCR products were separated on 1.5% agarose gel Numbers on the right side show the size of PCR products R, resistant; S, susceptible M1 is O'GeneRuler DNA Ladder Mix and M2 is GeneRuler™ DNA Ladder Low Range (Thermo Scientific) 40 Figure 4.11 PCR products of five selected lines showing size difference Detection of genes for resistance to Pm-0 and QTL of powdery mildew resistance by marker MR5 (primer pairs MR5-F and MR5R) on selected pumpkin lines PCR products were separated on 1.5% agarose gel Left image is electrophoresis after 25 min; picture must be electrophoresis after 40 minutes R, resistant; S, susceptible M1 is O'GeneRuler 41 Figure 4.12 Artificial infection of P xanthii fungus on Cucumis melon after infection 44 Figure 4.13 Detection of powdery mildew on some melon plants after infection 44 Figure 4.14 Disease severity of artificially inoculated Cucumis melon lines in plastic pots through seven times assessment The data were collected after 5days inoculation PM spores The yellow arrows represent lines with resistance The red arrows represent lines with potential resistance 49 vii Figure 4.15 Disease severity of artificially inoculated Cucumis melon lines in conditions of leaf disc The data were collected after days following inoculation Red arrows indicate lines carrying potential resistance gene 50 Figure 4.16 Indicator BSA12-LI3ECOR1 detects QTL Bpm12.1 resistance to P xanthii on Cucumis melon .50 Figure 4.17 PCR detection of QTL Bpm12.1 resistance to P xanthii by the indicator BSA12-LI3ECOR1 on selected lines of Melon Numbers on the right side show the size of PCR products R, resistant; S, susceptible M1 is O'GeneRuler DNA Ladder Mix and M2 is GeneRuler™ DNA Ladder Low Range (Thermo Scientific) .51 Figure 4.18 Characteristics of the PM4-dCAPS indicator closely associated with P xanthii resistance genes on Cucumis melo P1 is an R-resistant strain, P2 is an S-infected line (Yuste-Lisbona et al., 2011) 51 Figure 4.19 Detection of P xanthii resistance gene by PM4-dCAPS indicator on selected lines of Melon M2 is GeneRuler™ DNA Ladder Low Range (Thermo Scientific) 52 Figure 4.20 Artificial infection of P xanthii fungus on wax gourd after infection 55 Figure 4.21 Symptoms of selected wax gourd lines after three inoculation PM spores .55 Figure 4.22 Disease severity of artificially inoculated was gourd lines in conditions of leaf disc The data were collected after days following inoculation Red arrows indicate lines carrying potential resistance gene 58 Figure 4.23 Leaf disc samples of some wax gourd lines after 5days inoculation 59 Figure 4.24 Detection of P xanthii resistance gene by PM4-dCAPS indicator on selected zucchini lines M2 is GeneRuler™ DNA Ladder Low Range (Thermo Scientific) 61 Figure 4.25 PCR detection of QTL Bpm12.1 resistant to P xanthii by indicator BSA12-LI3ECOR1 on selected was gourd lines R, resistant; S, susceptible M1 is the O'GeneRuler DNA Ladder Mix and M2 is the GeneRuler™ DNA Ladder Low Range (Thermo Scientific) 62 Figure 2.26 Detection of Pm-0 resistance gene by marker S9_1539675 (primer pairs S9_1539 675-F and S9_1539 675-R) on selected squash lines viii M1 is O'GeneRuler DNA Ladder Mix (Thermo Scientific) A Yellow arrow indicates incompletely cleaved PCR product A blue arrow indicates PCR products of unknown origin .62 Figure 4.28 Symptoms of some selected cucumber lines after two inoculation PM spores 66 Figure 4.29 Disease severity of artificially inoculated cucumber lines in conditions of leaf disc The data were collected after days following inoculation Red arrows indicate lines carrying potential resistance gene 69 Figure 4.30 Leaf disc samples of some cucumber lines after 5days inoculation 69 ix 68 Figure 4.26 Disease severity of artificially inoculated cucumber lines in conditions of leaf disc The data were collected after days following inoculation Red arrows indicate lines carrying potential resistance gene 4.5.2 Evaluation of resistance to powdery mildew on Cucumber in leaf discs The result of resistance to powdery mildew on cucumber in leaf disc is revealed on the table 4.10 DC-8 and DC-11 showed no infection symptoms There were also low levels of infection on DC-5, DC-6 and DC-7 It can be seen that the results of assessing the tolerance of cucumbers in the leaf disc experiment were quite different from those in the pot experiment after the first days of infection In summary, DC -11 is considered to be the line with the highest potential disease resistance compared to the rest Figure 4.27 Leaf disc samples of some cucumber lines after 5days inoculation 69 Table 4.10 Disease severity result of artificially inoculated cucumber gourd lines in conditions of leaf disc The data were collected after days after inoculation Line N Average scale SE DC-1 1.40 0.25 DC-2 1.40 0.25 DC-3 1.40 0.40 DC-4 1.25 0.36 DC-5 1.00 0.00 DC-6 0.80 0.20 DC-7 0.80 0.20 DC-8 0.00 0.00 DC-9 1.40 0.25 DC-10 1.40 0.25 DC-11 0.00 0.00 70 V CONCLUSION 5.1 CONCLUSION 5.1.1 Identification of powdery mildew fungus on cucurbits Morphological and molecular characterization identified the fungus causing powdery mildew of pumpkin collected in Gia Lam being Podosphaera xanthii This is for the first time this species identified to cause powdery mildew on cucurbits in Vietnam 5.1.2 Evaluation of pumpkin resistance All 30 pumpkin lines were susceptible to powdery mildew with an average disease severity of (highest) at the 7th evaluation The S9_1539 675 marker is suitable for detecting the resistant gene Pm0 in pumpkin All 30 pumpkin lines are homozygous for the Pm-0 resistant gene and not have QTL resistance derived from Cucurbita okeechobeensis subsp martinezii 5.1.3 Evaluation of melon resistance 23/24 lines of melon were infected P xanthii with an average disease severity of 4.2 -8 at the seventh evaluation The melon line DL-4 is completely free of infection through seven evaluations (Resistance) The DL-25 was mildly infected with an average disease severity of 4.2 at the 7th assessment (potential resistance) Two markers BSA12-LI3ECORI (InDel CAPS) and PM4-dCAPS (CAPS) did not detect the resistant genes linked to these two marker from lines DL-4 and DL25 5.1.3 Evaluation of wax gourd resistance 30/30 lines of wax gourd were not infected or very lightly infected with powdery mildew The lines of BX-11, BX-26 and BX-30 were mildly infected 71 with an average disease severity of less than 1.5 at the last evaluation All chosen markers for the genus Cucurbita and the genus Cucumis were not suitable for detecting resistance genes on wax gourd 5.1.4 Evaluation of cucumber resistance 10 Four of eleven cucumber lines showed potential resistance to powdery mildew disease Of them, the DC-11 was considered to be the line with the highest potential disease resistance compared to the rest The DC-5, DC-6 and DC-7 lines had milder infection symptom 5.2 RECOMMENDATION Continuing to evaluate the infection resistance on wax gourd Optimizing PCR reaction conditions for the indicators on Melon, namely BSA12-LI3ECORI (InDel CAPS) and PM4-dCAPS (CAPS) 72 REFERENCE Barghamdi B, Ghorat F, Asadollahi K, Sayehmiri K, Peyghambari R, Abangah G 2016 Therapeutic 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Nile Valley Benincasa hispida Wax gourd Southeast Asia, Southeast Asia Australia Cucumis melosubsp Melo Melon (numerous names) India and Australia Asia India Asia, East Asia New World Africa Asia and Melanesia Cucumber, Cucumis sativus xishuangbanna cucumber 79 Center of No Domestication 10 Species Lagenaria siceraria Common names Bottle gourd Momordicachar Bitter gourd antia “minor crops” Benincasa Tinda fistulosa Citrullus Preserving amarus desert melon Citrullus colocynthis Colocynth Area of origins Africa Africa Area of domestication Independently domesticated in Eurasia and south America Africa and India India (?) India, Pakistan, East Africa South Africa Mediterranean West Africa West Africa, Middle East to India, Northern Africa Citrullus Egusi Melon East Africa East Africa mucosospermus Coccinia India and Scarlet gourd East Africa grandis Southeast Asia Africa, Cucumis Maroon naturalized Tropical anguria cucumber in the New regions World Cucumis melo Tibish and Africa Sudan subsp Meloides Fadasi melon Cucumis Kiwano Sub-Sahara World wide metuliferus cucumber Africa Cyclanthera Stuffing South South America pedata cucumber America 80 Center of No Domestication Species Common names Area of origins Area of domestication 10 Hodgsonia macrocarpa Lard fruit Himalaya China 11 Luffa acutangula Arabian Sponge gourd, Peninsula, angled gourd India Tropical regions 12 Luffa aegyptiaca Sponge gourd Southeast Asia Tropical regions Egusi gourd Central America West Africa, Central and South America Mouse melon, Mexico mininature watermelon Central America Central America Melothria mannii 13 (syn.Cucumerop sis mannii) 14 Melothria scabra 15 Momordia dioica Spiny gourd South Asia South Asia (especially India), Australia 16 Momordica balsamina Balsam apple Tropical Africa Mediterranean (?) 17 Momordica cochinchinensis 18 Sicana odorifera Gac Cassbanana 81 Southeast Asia to Southeast Asia North Australia South America Latin America ans Southern United States Center of No Domestication Common names Species Area of origins Area of domestication Chouchou Mexico Tropical regions Luo Han Guo Southern China, Northern Thailand China Fluted gourd Tropical West Africa Tropical West Africa “Minor Crops” 19 Sicyos edulis (Sechium edule) 20 Siraitia grosvenorii 21 Telfairia occidentalis 22 Telfairia pedata 23 Trichosanthes cucumerina Oysternut, Zanzibar oil vine Snake gourd 82 Tropical Tropical East East Africa Africa Southeast Asia(?) China