Isolation and characterization of fungus associated with leaf spot of grapevines in viet nam (khóa luận tốt nghiệp)

However, recently many diseases appeared and affected the yield and quality such as downy mildew Plasmopara viticola, powdery mildew Uncinula necator, and leaf spot Phomopsis viticola..

GRAPEVINES IN VIET NAM

Student’s name : Giap Thi Thuy Chi

Student’s code : 620429

Class : K62-CNSHE

Supervisor : Dr PHAM HONG HIEN

MSc NGUYEN THANH HUYEN

HA NOI – 2022

Giap Thi Thuy Chi

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ACKNOWLEDGEMENTS

First and foremost, I have to thank the Vietnam National University of Agriculture for giving me a chance to study in a professional environment that provided me the sufficient knowledge throughout four and a half academic years In doing my thesis, many people helped me bring this work into existence, but the most influential and inspiring person I would like to extend thanks to is my supervisor, MSc Nguyen Thanh Huyen without her assistance and dedicated involvement in every step throughout the thesis, this thesis would have never been accomplished

I would like to express my deep gratitude to all teachers from the Faculty

of Biotechnology, who encouraged me to enhance my knowledge and help me get orientation for my thesis

Getting through my undergraduate thesis required more than academic support, and I have many, many people to thank for listening to and, at times, having to tolerate me, thank all my friends, who were with me up until this point, simultaneously motivate me to accomplish more

Last but not least, I would like to thank myself, and my parents who endured this long process, for always supporting and assisting me during the process of completed graduate thesis

Ha Noi, May, 2022 Student

Giap Thi Thuy Chi

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ABSTRACT

Grapevine (Vitis spp.) is among the most widely grown fruit crops

worldwide and plays a significant role in its commercial value in Vietnam as well as having an excellent source of nutrition However, recently many diseases

appeared and affected the yield and quality such as downy mildew (Plasmopara viticola), powdery mildew (Uncinula necator), and leaf spot (Phomopsis viticola) Among them, leaf spot disease is a fairly common disease and causes a

significant reduction in the yield of Grapevines

The thesis was conducted in leaf spots of grapevines collected from different places in Ha Noi and Bac Giang, Vietnam in 2021 The fungus was Isolated from leaf spot disease on grapevines, and the fungus was selected and then used for artificial infection into healthy grapevine leaves The fungal strains causing the grape leaf spots will be selected to study the morphological and biochemical characteristics, as well as the conditions affecting the growth of the fungus Then, these fungal strains are identified by molecular tools

Research results show that 2 strains (SM and HV) of fungi have been isolated, but after re-infection, only one strain (SM) is capable of causing leaf spot disease on grape leaves, SM strain has mycelium that is profusely branched (monopodial branching)

Studying the growth conditions of fungal strains proves that SM grows well on MEA and PDA, at around the temperature range of 20-30°C with pH from 5.0 to 8.0 SM strain has the ability to produce chitinase, cellulase, and pectinase

The causal fungus strain (SM) was identified as Phomopsis viticola

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INDEX

COMMITMENT i

ABSTRACT iii

LIST OF ABBREVIATIONS vi

LIST OF TABLES vii

LIST OF FIGURES viii

I INTRODUCTION 1

1.1 Introduction 1

1.2 Research purpose 2

1.3 Requirement 2

II LITERATURE REVIEW 2

2.1 General introduction of grapevines 2

2.1.1 Distribution 4

2.1.2 Description of morphological characteristics of the grapevine 5

2.1.3 Nutrients 6

2.1.4 Uses 7

2.2 Grapevine pathogens 8

2.2.1 Downy mildew (Plasmopara viticola) 8

2.2.2 Powdery mildew (Uncinula necator) 10

2.2.3 Leaf spot (Phomopsis viticola) 11

2.3 Commercial production 14

III MATERIALS AND METHODS 17

3.1 Materials 17

3.1.1 Sample collection 17

3.1.2 Chemicals and medium components 17

3.1.3 Laboratory apparatuses and equipment 19

3.1.4 Research location and time 19

3.2 Methods 19

3.2.1 Isolation of fungus causing leaf spot disease on grapes (Chomnunti et al ,2014) 19

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3.2.2 Artificial infection (Jayawardena, Ruvishika S., et al.,2016) 19

2011) 20

3.2.4 The effects of some factors on fungal growth (culture medium, temperature, and pH ) 20

3.2.5 Extracellular enzyme production of isolated fungal strain 21

3.2.6 DNA extraction, PCR amplification and Sequencing 22

3.2.7 Phylogenetic analysis 23

IV RESULTS AND DISCUSSION 24

4.1 Isolation of fungal pathogens causing leaf spots from grapevines leaf 24

4.2 Artificial infection 25

4.3 Morphological characteristics of SM strain 25

4.4 The effects of culture medium on SM strain growth 27

4.5 Effect of different temperatures on the growth of SM strain 28

4.6 Effect of different pH on the growth of SM strain 29

4.7 Testing extracellular enzyme activity 30

4.8 DNA extraction, PCR amplification and Sequencing and Phylogenetic analysis 33

4.8.1 DNA extraction, PCR amplification of SM strain 33

4.8.2 Phylogenetic analysis of SM strain 34

V CONCLUSION AND SUGGESTION 36

5.1 Conclusion 36

5.2 Suggestion 36

REFERENCES 37

vi

LIST OF ABBREVIATIONS Abbreviations Explanations

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Figure 2.1 One grapevine garden in Ninh Thuan 5

Figure 2.2 Symptoms of powny mildew disease 9

Figure 2.3 Symptoms of powdery mildew disease 11

Figure 2.4 Symptoms of leaf spot disease 13

Figure 3.1 Leaf samples with leaf spot disease 17

Figure 4.1 Fungal strains isolated from infected grape leaves 24

Figure 4.2 Re-infection of isolated fungal strains (HV, SM) in grapevine leaf and control sample on the 3rd days 25

Figure 4.3 Microscopic observation of the selected fungus strain-SM 26

Figure 4.4 Growth of SM strain in different media on the 5th days 27

Figure 4.5 Growth of SM strain in different temperatures on the 5th days 28

Figure 4.6 Growth of SM strain in different pH on the 5th days 30

Figure 4.7 Chitinase activity of the SM strain 31

Figure 4.8 Cellulase activity of the SM strain 32

Figure 4.9 Pectinase activity of the SM strain 32

Figure 4.10 PCR product of SM strain on gel agarose 33

Figure 4.11 Phylogenetic tree of SM strain 34

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I INTRODUCTION 1.1 Introduction

Grapevine (Vitis sp., Vitaceae) is one of the most important fruit crops

cultivated around the globe and has a high nutritional value in many countries According to FAO data (2014), the global grape-growing area has 8,485,000 hectares and is increasing at a pace of 2% per year, with yearly production reaching 60,473 million tons, accounting for 17.5 percent of total global fruit production Approximately 71% of grapes are utilized as raw materials for wine production, 27% are consumed fresh, and 2% are dried (seedless grapes)

Currently, vines are grown on all five continents in climates that are conducive to their growth Spain (1,175,000 ha), Russia, Italy, France, Turkey, Portugal, and the United States are the countries with the largest grape areas in the world Over 60% of the world's grape output has been produced in Europe

In recent years, grape acreage and production have expanded in Asia The entire area is currently around 1.7 million hectares China, India, Japan, Korea, Pakistan, Thailand, and Vietnam are Asian countries having substantial grape acreage (FAO, 2014)

According to the conference on grape production in Asia Pacific countries, which included eight countries: China, India, Korea, Japan, Myanmar, Thailand, and Vietnam, the grape area of the participating countries

is 393,725 ha, with a yield of 5.432 million tons and an average grape yield of 13.6 tons per ha per year The grape output in Vietnam is lower than the regional average, at only 11.2 tons per hectare per year (caytrong, 2019)

In Viet Nam, there are also many grape growing regions, of which Ninh Thuan

is the most planted with about 2,400 hectares in Ninh Thuan (accounting for 90%) and 250 hectares in Binh Thuan (accounting for 9% of the total grape

growing area of the country) (Le Quang Quyen et al., 2019) The results of a

random survey of 50 vineyards with different planting ages in 2014 showed that

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the average grape yield in Ninh Thuan was over 30tons/ha/year Growing

grapes can make a profit of over 200-300 million VND/ha/year (Tran Ngoc Tu

et al., 2015)

Although it has great economic value, currently the yield and quality of

vines are reduced due to several diseases such as downy mildew (Plasmopara viticola (Berk & MA Curtis) Berl & De), anthracnose (Elsinoe ampelina Shear), leaf spots (Phomopsis viticola) and Cooked Rot (Glomerella cingulata (Stoneman) Spauld & Schrenk) Currently, there are many measures to control

diseases on grapevines, but measures Using chemical drugs are widely applied because of their quick effect However, the long-term use of chemical drugs causes serious health consequences

For the above reasons, the topic " Isolation and characterization of fungus associated with leaf spot of grapevines in Viet Nam " was carried out This study was undertaken for the evaluation of the characteristics of pathogenic fungal strains and will provide useful information to find solutions Effective disease prevention while maintaining the nutritional value of grapes without affecting the health of the user

1.2 Research purpose

- Isolation of fungal strains causing leaf spot disease on grapes in Vietnam

- Determination of the morphological, and biochemical characteristics of

the pathogenic fungus causing grape leaf spot spots Vietnam

1.3 Requirement

- Isolation and selection of fungus associated with leaf spots in grapevine leaves

- Pathogenicity test on healthy grapevine leaves

- Characterization of fungal strain causing leaf spot in grapevine leaves: morphology colony, mycelium, and spore of fungus

America, and Europe under subtropical, Mediterranean, and continental–

temperate climatic conditions It is the single Vitis species that has gained

significant economic interest and is a commercially valuable plant over time,

which can be used as a rich source of nutrients in multiple industries (Xu et al 2017; Chitarrini et al., 2017), for example, the North American, are used as

breeding rootstock due to their resistance against grapevine pathogens, such as Phylloxera, Oidium and mildews Indeed, a great majority of cultivars are widely cultivated for fruit, juice, and mainly for wine which was classified as

Vitis vinifera L subsp Vinifera (or Sativa), derive from wild forms (Vitis vinifera L subsp sylvestris (Gmelin) Hegi] ((Rossetto, McNally et al., 2002); (Sefc, Steinkellner et al., 2003); (Crespan, 2004, This, Jung et al., 2004)

According to Nikolai Ivanovich Vavilov (1926), a Russian botanist work

of prominent, the highest biodiversity of a crop is usually its center of origin In

1938, his disciple Aleksandr Mikhailovich Negrul observed that the highest biodiversity in cultivated grapes was in today’s Georgia, Armenia, Azerbaijan, Turkey, and Iran, which makes them the best candidates for being the cradle(s)

of viticulture

Analysis of morphological similarities between wild and cultivated grapes from all over Eurasia generally supports a Near Eastern origin for grape domestication The closest genetic relationship was discovered between local wild grapevines and traditionally cultivated grape varieties from southern Anatolia, Armenia, and Georgia This suggests that the headwaters of the Tigris and Euphrates rivers in the Taurus Mountains are the most likely location for the first domestication of the grapevine (Arroyo‐García, R., et al., 2006)

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Grapevine was domesticated (Vitis vinifera, sometimes called V sativa)

and was one of the most important fruit species in the classic Mediterranean world, and it is the most important economic fruit species in the modern world today As in the ancient past, sun-loving grapevines are today cultivated to produce fruits, which are eaten fresh (as table grapes) or dried (as raisins), and, most especially, to make wine, a drink of great economic, cultural, and symbolic value (K Kris Hirst, 2018)

The Vitis family consists of about 60 inter-fertile species that exist almost exclusively in the Northern Hemisphere: of those, V vinifera is the only one

extensively used in the global wine industry Approximately 10,000 cultivars of

V vinifera exist today, although the market for wine production is dominated by

only a handful of them Cultivars are typically classified according to whether they produce wine grapes, table grapes, or raisins (K Kris Hirst, 2018)

Domesticated grapevine (Vitis vinifera, also known as V sativa) was one

of the most important fruit species in the classic Mediterranean world, and it continues to be the most important economic fruit species in the modern world Sun-loving grapevines are cultivated today, as they were in the past, to produce fruits that are eaten fresh (as table grapes) or dried (as raisins), and, most importantly, to make wine, a drink with significant economic, cultural, and symbolic value (K Kris Hirst, 2018)

The Vitis family consists of approximately 60 inter-fertile species that exist almost entirely in the Northern Hemisphere; of these, V vinifera is the only

one that is widely used in the global wine industry There are approximately

10,000 V vinifera cultivars in existence today, but only a few of them dominate

the wine production market Cultivars are typically classified based on whether they produce wine grapes, table grapes, or raisins (K Kris Hirst, 2018)

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2.1.1 Distribution

Currently, in the world, the area under grape cultivation reaches 7.4 million hectares (M ha) Spain is the biggest country with 969 thousand hectares (k ha), followed by China (875 k ha), France (789 k ha), and Italy (702 k ha) (OIV, 2018)

In Asia, the area and production of grapes have increased in recent years Currently, the total area is about 1.7 million hectares The Asian countries with significant grape acreage are China, India, Japan, Korea, Pakistan, Thailand, and Vietnam According to the conference on grape production in Asia Pacific countries including 8 participating countries: China, India, Korea, Japan, Myanmar, Thailand, and Vietnam, the grape area of the participating countries is 393,725 ha, yield 5.432 million tons, average grape yield is 13.6 tons/ha/year Vietnam's grape yield is lower than the regional average, reaching only 11.2 tons/ha/year The region from North Binh Thuan to South Khanh Hoa, especially Ninh Thuan province, is located in the driest region in the country, with a lot of wind, low humidity (average 70-75%), low rainfall (<800mm/year) This is a favorable condition for vines to grow and develop well As a result, in Ninh Thuan, grapes soon became commodity and specialty crops from the mid-80s of the last century It plays an important role in agriculture and the province's economy The south-central province, which has the least rainfall in the country, has more than 1,200 ha of grape growing areas with an annual total output of 30,000 tonnes, with the red cardinal and the NH01-48 greencardinala the two major grape varieties The province’s grape farming area accounts for less than 4% of its farming area, but the revenue contributes 15-20% of its total crop revenue, according to the Department of Agriculture and Rural Development

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Figure 2.1 One grapevine garden in Ninh Thuan

(source: Báo nông nghiệp Việt Nam)

The vineyards produce large berries with black, red, purple, and blue colors and are a specialty of Ninh Thuan The province's grape growing area is about 2,500 hectares, concentrated mainly in Ninh Phuoc and Ninh Hai districts and Phan Rang - Thap Cham town, in communes and towns of Phuoc Dan, Phuoc Huu, and Phuoc Son with many new grape varieties Since 2002, Ninh Thuan has expanded the vine area from 25 to 30 hectares and since 1995 the area has increased fastest to over 2,000 hectares The locality has a plan to develop vines from 1,000ha -2,000ha in 2015 and 2,500ha in 2020

Ninh Thuan is a supplier of fresh grapes with the largest production in the country, averaging about 15,000 tons/year and processing many products such

as dried grapes, grape jam, grape wine, grape nectar of consumers in which, every year farmers of Ninh Thuan province, export over 10,000 tons of fresh grapes to other provinces in the region

2.1.2 Description of morphological characteristics of the grapevine

The grapevine (Vitis vinifera L.) is a vine cultivated worldwide for its

edible berries (grapes) that are eaten fresh or pressed to make wine or grape juice An important by-product of the grape crop is the leaves and shoots that are

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pruned once a year for crop management Grape leaves and vine shoots have been used traditionally to feed livestock in vineyards, particularly in Mediterranean countries

The grapevine is a vigorous and climbing woody vine that can reach a height of 15-20 m in the wild In cultivated vines development is dramatically reduced through yearly pruning of shoots and leaves Grape leaves are simple circular or circular ovate in outline, 5-25 cm broad, with long petioles They can

be entire or lobed (3 to 5 lobes) and coarsely toothed The inflorescence bears small, pentamerous, fragrant, yellow-green flowers The fruits are ellipsoid or globose berries, 6-25 mm long, of variable colors (from green, yellow to dark blue-purple) depending on the variety Fruits (grapes) are juicy, sweet ,or sour They contain 3-4 seeds (Edward W Hellman, 2017) In viticulture, the shoot is the vegetative stem of the current year that bears the leaves, tendrils, and fruit The cane is the vine shoot from the period it matures (turns brown and woody) until the end of the second year of growth (Jackson, 2008)

2.1.3 Nutrients

Grapes, particularly the Concord grape variety, contain phytonutrients such as phenols, polyphenols, resveratrol, and carotenoids Grapes are also a good source of vitamin K, copper, and many B vitamins, adding that they are also high in fiber and low in calories

Table 2.1 Nutrition of Grapes Amt per Serving %DV Amt per serving %DV

Protein 1g

( Source: Candy Hearts® Grapes used with permission from IFC )

1 cup (151 g) Calories 104 Calories from Fat 2; Percent Daily Values (%DV) are based on a

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2,000calorie diet.

Grapes contain 13 calories per 14 g serving This serving contains 0.3 g of fat, 0.8 g of protein, and 2.4 g of carbohydrates The latter is 0.9 g of sugar and 1.5 g of dietary fiber; the rest is complex carbohydrates Grapes contain 0 g of saturated fat and 0 mg of cholesterol per serving 14 g of raw grape leaves contains IU vitamin A, 1.6 mg vitamin C, and 0.00 mcg vitamin D, as well as 0.37 mg iron, 50.82 mg calcium, and 38 mg potassium

2.1.4 Uses

In vineyards, grape leaves and shoots are pruned after the grapes are harvested These residues are generally disposed of by mulching into the vineyard or by burning on-site (Letaief, 2016) In the Mediterranean Basin, leaves and shoots are traditionally browsed by sheep and goats (Magnier, 1991)

In Turkey, grape leaves are an important source of forage for ruminants during the critical period when the quality and quantity of pastaree is limited (Kamalak, 2005) Ensiled grape crop residues have been recommended as fodder since the end of the 19th century (Pau, 1940) Generally, grape leaves and shoots are fibrous products that can only be eaten by ruminants, equids, and rabbits Grape leaves are also used for food in many countries in the Mediterranean area, Eastern Europe, and Asia Grape leaves stuffed with rice are delicacies known as

"yaprak dolma" in Turkey, "dolmeh barg mo" in Iran, and "warak enab" in

Egypt and Lebanon (Bekhit et al., 2016) Grape leaves and shoots have been

reported as a potential source of natural antioxidants and as a food supplement

(Cetin et al., 2011)

The grape is one of the world's most important fruit crops in the area, production and value In 2012, grapes were cultivated on more than 7 million ha and produced 69 million t of fruits, with an average yield of 9.6 t/ha 40 million t

of grapes were used for the production of wine (about 90%) and juice (around 10%, fresh or concentrated), 24 million t for fresh fruit, and 5 million t for dried

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grapes (raisins) (OIV, 2016; Castellucci et al., 2013) The most important

producers were China (9.6 million t), the USA (6.6 million t), Italy (5.8 million t), France (5.3 million t), Spain (5.2 million t), Turkey (4.2 million t), Chile (3.2 million t), Argentina (2.8 million t) and Iran (2.1 million t) The EU represents 75% of grape production and 57% of wine production The main wine producers are Italy, France, Spain (where most of grape production is used for wine) and the USA China is mostly a producer of fresh fruits (FAO, 2016)

2.2 Grapevine pathogens

For farmers, grapevine diseases can be devastating to the productivity and quality of vines Unfortunately, many kinds of vine diseases thrive in all sorts of conditions Many of the grapevine parasites are of animal origin (insects and

mites) or vegetal origin (cryptogram or parasitic fungi) The most common fungal diseases are downy and powdery mildew (Plasmopara viticola and Uncinula necator), and leaf spot (Phomopsis viticola) On the other hand, the most dangerous insects are the grape moth (Lobesia botrana), vine mealybug (Planococcus ficus), and the citrus mealybug (Planococcus citri)

2.2.1 Downy mildew (Plasmopara viticola)

The downy mildew was introduced in France from North America in

1878 It moved into Italy the following year and subsequently into the other countries of the Mediterranean basin Furthermore, it was introduced into Australia in 1919 and into New Zealand in 1926

The symptoms of this pathogen differ somewhat on different hosts However, they all cause yellow spots on the upper leaf surface between the leaf veins These spots spread everywhere but the veins and eventually turn brown The plant cannot photosynthesize on these yellow or brown spots When the leaf

is totally brown, it drops Today downy mildew is present in all vine cultivation areas The downy mildew is the most prevalent form of mildew and is usually

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spread by rainfall It attacks leaves, shoots, and berries and can quickly defoliate the vine leading to the loss of the entire crop When the leaf is totally brown, it drops If the plant loses too many leaves, the plant dies Optimum conditions for primary infection take place at 10:10:10, which corresponds to at least 10 mm of rain at a temperature of 10oC or more, over 10 h

Figure 2.2 Symptoms of powny mildew disease

(source:

https://vikaspedia.in/agriculture/crop-production/integrated-pest-managment/ipm-for-fruit-crops/ipm-strategies-for-grapes/grapes-diseases-and-symptoms)

Fungus (Plasmopara viticola), the causal agent of grapevine downy

mildew, is a heterothallic oomycete that overwinters as oospores in leaf litter and soil In the spring, oospores germinate to produce macrosporangia, which under wet conditions release zoospores Zoospores are splashed by rain into the canopy, where they swim to and infect through stomata After 7–10 days, yellow lesions appear on the foliage During favorable weather, the lesions sporulate and new secondary infections occur

Downy mildew can be controlled by spraying various chemicals either as preinfection or postinfection treatments There are two groups of spray chemicals, those with a single-site activity that acts on only one site within the fungus organism, or those with multisite activity, which work on more than one site within the fungus The most used multisite chemicals for the preventive

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control of downy mildew are copper salts such as copper oxychloride In the past several years, the overuse of chemicals has led to small mutational changes within the fungus, which can lead to the fungus being resistant

Downy mildew is currently causing heavy damage to grape yield and the use of some drugs such as metiram, mancozeb, folpet, tolylfluanide are commonly used to combat Downy mildew is becoming more and more

common

2.2.2 Powdery mildew (Uncinula necator)

The powdery mildew is a pathogen that was brought into England from North America in 1845 Subsequently, powdery mildew was introduced into France in 1847, Belgium in 1848, and finally Italy in 1849

Symptoms usually appear later in the growing season on outdoor plants Powdery mildew starts on young leaves as raised blister-like areas that cause leaves to curl, exposing the lower leaf surface Infected leaves become covered with a white to gray powdery growth, usually on the upper surface; unopened flower buds may be white with mildew and may never open Leaves of severely infected plants turn brown and drop The disease prefers young, succulent growth; mature leaves are usually not affected All aerial parts of the plants can get infected by this disease Development happens through the entire growing season until late fall It favors dry years and climates with low humidity and little rainfall Moldy coats on leaves, inflorescences, and rachis are telltale signs Berries can become infected in the early stages of their development, with a gray coat covering them and then they dry out afterward Infected half-ripe berries usually crack and dry out The plant’s canes can also become covered with a gray coating with gray-brown spots underneath

a pin

To control this pathogen, genetic resistance, chemical methods, and careful farming are used to manage conditions Fungicides are used as well as silicon, which assists plant cells in fighting against the fungus and strengthening the epidermal cells of the plants There are no approved fungicides for postinfection treatments

2.2.3 Leaf spot (Phomopsis viticola)

Leaf spots are a type of plant disease that is usually caused by fungus and sometimes other cases such as herbicide injuries Leaf spots can vary in size,

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shape and colours depending on the age and type of the cause or pathogen Grapevines are weakened by the spots on the leaves as they reduce available foliar space for photosynthesis

The causes of leaf spots are mainly Phomopsis viticola (Sacc.) Sacc is historically known to infect grapevines (Vitis vinifera and Vitis spp.) causing

leaf spot disease However, leaf spots may also be caused by abiotic factors such

as environmental conditions, toxicities and herbicide injuries (Isleib, 2012)

Leaf symptoms appear early in the growing season and are characterized

by small brown necrotic spots around the veins and brown “U- or V-” shaped lesions at the margins of the leaf blade Under humid conditions, abundant gray sporulation can be observed on the underside of the diseased leaf tissues to black necrotic irregular-shaped lesions, and clusters show rachis necrosis and brown, shriveled berries close to harvest (Hewitt, W B., and Pearson, R C , 1988)

Leaf spot is a well-studied grapevine disease, and much has been written about its etiology (Mostert, L., Crous, P W., Kang, J C., and Phillips, A J L , 2001), epidemiology (Erincik, O., Madden, L V., Ferree, D C., and Ellis, M A., 2003.)

Certain chemicals are used to treat leaf spot disease, such as the Bordeaux mixture, the first fungicide to have been developed, which treats many fungal and bacterial leaf spots (Agrios, G N., 2005) Other fungicides such as zineb, chlorothalonil and Captan, also treat leaf spot disease and Benomyl specifically treats Cercospora leaf spots, cherry leaf spot and black spot of roses(Agrios, G

N , 2005)

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Figure 2.4 Symptoms of leaf spot disease

A Grape leaf spot symptoms, B conidia with conidiophores of Phomopsis viticola, C

conidia Phomopsis viticola

(Schilder, Erincik et al., 2005) Some other studies also show that Phomopsis sp

is the causative agent of leaf spot disease: (Merrin, Nair et al., 1995) published a study on Phomopsis from grapevines in Australia Phomopsis was also isolated from grapes (Vitis spp.) showing symptoms of leaf spot in Ohio, Michigan, New York, Pennsylvania, Maryland, and Ontario (Canada) (Schilder, Erincik et al., 2005) Severe rachis and berry infections were observed in a 'Vignoles' (Vitis

interspecific hybrid) vineyard in southwestern Michigan in 1998 and 1999 Besides, During the 2002 to 2004 growing seasons, a statewide survey for the

incidence of leaf spot of grape (caused by Phomopsis viticola) was conducted

within vine on disease incidence Disease was observed in all surveyed

about the specific effects of the environment on Phomopsis leaf spot

development Disease incidence and severity have been reported to be higher in

wet growing seasons than in dry ones (Willison, Chamberlain et al., 1965)

Prolonged rainy weather, particularly in early spring when new shoots emerge,

has been found to be very conducive to infection (Schilder, Erincik et al., 2005)

linked early-season infections to low (presumably below-normal) temperatures during this time period They claimed that the fungus was only active at low temperatures and that as temperatures rose, the fungus became inactive, resulting in no infection later in the growing season Bugaret (1984) was the first

to explore the effect of temperature and wetness duration on foliar infection by

Phomopsis viticola He stated that the ideal temperature for infection was

between 23 and 25°C He also reported that the average temperature at bud break in the spring ranged from 8 to 18 °C, which is significantly lower than the optimum observed in his studies He also confirmed that at the optimum temperature, infection requires at least 4 hours of wetness

2.3 Commercial production

Like the rest of the global agricultural sector, the table grape industry has had to face a variety of challenges related to the COVID-19 pandemic this past year, including soaring freight costs, labor shortfalls, worldwide shipping delays and refrigerated container shortages In Peru, worker strikes hampered then in Chile, unseasonal downpours in January led to losses of up to 80% in the O’Higgins Region, the country’s largest grape production areimilar to , the

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blockage of the Suez Canal in March created a substantial traffic jam on both sides, bringing Indian grape exports destined for Europe to a standstill However, despite these obstacles, the table grape industry has experienced a successful season, with very little impact on the global table grape output in marketing year 2020/21 According to a recent report by the U.S Department of Agriculture’s Foreign Agricultural Service, global table grape production in 2020/21 is estimated to reach approximately 24.7 million metric tons, just below the record volume of 25.0 million tons achieved in 2017/18 On the back of this plentiful supply, table grape exports in 2020/21 are forecast to surge to a record high in excess of 3.4 million tons Record high in excess of 3.4 million tons

Figure 2.5 Import volume of grapes worldwide in 2020/2021, by leading

countries

(Source: https://www.statista.com/statistics/756476/global-top-grape-importer-worldwide/)