SURVEILLANCE AND PATHOGEN CHARACTERIZATION
OF BACTERIAL CANKER OF STONE FRUITS
RAEES AHMED
10-arid-1770
Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018 2
SURVEILLANCE AND PATHOGEN CHARACTERIZATION
OF BACTERIAL CANKER OF STONE FRUITS
by
RAEES AHMED
(10-arid-1770)
A thesis submitted in partial fulfillment of
the requirement for the degree of
Doctor of Philosophy
in
Plant Pathology
Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018
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CONTENTS
Page
List of Tables ix
List of Figures x
List of Appendices xiv
List of Abbreviations Xv
Acknowledgements Xvii
ABSTRACT xix
1. INTRODUCTION 1
1.1 STONE FRUITS 1
1.2 NUTRITIONAL VALUE OF STONE FRUITS 3
1.3 STONE FRUITS IN WORLD 3
1.4 STONE FRUITS IN PAKISTAN 4
1.5 DISEASES OF STONE FRUITS 6
1.6 BACTERIAL CANKER OF STONE FRUITS 7
2. REVIEW OF LITERATURE 13
2.1 BACTERIAL CANKER OF STONE FRUITS 13
2.2 BIOCHEMICAL CHARACTERIZATION 14
2.3 MOLECULAR CHARACTERIZATION 15
3. MATERIALS AND METHODS 17
3.1 COLLECTION OF SAMPLES 17
3.2 DETERMINATION OF DISEASE INCIDENCE AND 17
DISEASE PREVALENCE
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3.3 ISOLATION AND PURIFICATION OF PATHOGENIC 18
BACTERIA FROM DISEASES SAMPLES
3.4 INITIAL SCREENING OF BACTERIAL CULTURES 18
3.4.1 Gram Staining 18
3.4.2 KOH (Loop) Test 19
3.4.3 Florescent Under UV Light 19
3.5 PRESERVATION OF CULTURE 19
3.5.1 Preservation in Glycerol 19
3.5.2 Preservation in Mineral Oil 20
3.6 PATHOGENICITY TESTS 20
3.7 SYRINGOMYCIN BIOASSAY 20
3.8 BIOCHEMICAL CHARACTERIZATION OF BACTERIAL 21
CANKER PATHOGENS
3.8.1 Levan Production Test 21
3.8.2 Oxidase Test 21
3.8.3 Potato Soft Rot Test 22
3.8.4 Arginine Dihydrolase Test 22
3.8.5 Hypersensitivity Test 22
3.8.6 Gelatine Hydrolysis Test 23
3.8.7 Aesculin Hydrolysis Test 23
3.8.8 Tyrosinase Activity 23
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3.8.9 Tartrate Utilization Test 23
3.8.10 L-lactate Utilization Test 24
3.9 MOLECULAR CHARACTERIZATION AND GENETIC 24
VARIABILITY
3.9.1 Primers Used in Study 24
3.9.2 Extraction of Genomic DNA 24
3.9.3 Plasmid Extraction 25
3.9.4 PCR Amplification 26
3.9.4.1 PCR for 16S rRNA 26
3.9.4.2 PCR for virB1 and virD4 Genes 27
3.9.4.3 PCR for gyrB Gene 27
3.9.5 PCR Purification 28
3.9.6 DNA Clean and Concentration 29
3.9.7 Quantification of Purified PCR Product 29
3.9.8 Sequencing and Phylogenetic Analysis 29
4 RESULTS AND DISCUSSION 32
4.1 SURVEYS FOR DISEASE INCIDENCE 32
4.2 DISEASE INCIDENCE AND DISEASE PREVALENCE OF 32
BACTERIAL CANKER OF STONE FRUITS
4.2.1 Punjab Province 32
4.2.2 KPK Province 37
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4.3 ISOLATION AND PURIFICATION 48
4.4 INITIAL SCREENING OF BACTERIAL ISOLATES 48
4.5 PATHOGENICITY TEST 54
4.6 SYRINGOMYCIN BIOASSAY 58
4.7 BIOCHEMICAL CHARACTERIZATION OF 59
PATHOGENIC ISOLATES
4.8 MOLECULAR CHARACTERIZATION 71
4..8.1 16S rRNA Gene 71
4.8.2 gyrB Gene 72
4.8.3 virB1 and virD4 Gene 73
SUMMARY 82
CONCLUSIONS 85
FUTURE DIRECTIONS 86
LITERATURE CITED 87
APPENDICES 96
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List of Tables
Table No. Page
1 Nutritional values of some stone fruits 8
2 According to FAOSTAT, 2014 stone fruits production tons 8
3 According to FAOSTAT, 2014 stone fruits production area in hectares 8
4 Primer sequence used for PCR analysis 31
5 Screened isolates with their labeled name, their host and isolation 64
source
6 Pathogenicity of bacterial isolates on fresh peach fruits using pin 69
prick method
7 Syringomycin bioassay of pathogenic bacterial isolates 75
8 LOPAT scheme for the identification of P. syringae according to 79
Lelliott and Stead (1987)
9 GATTa and Lactate test for the confirmation of pathovars in P. 82
syringae according to Lelliott and Stead (1987)
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List of Figures
Figure No. Page
1 Map of Pakistan showing Khyber-Pakhtunkhwa, Punjab 9 and Baluchistan Provinces
2 Map showing districts of Province Baluchistan, Pakistan 9 3 Map showing districts of KPK Province and Northern 10
areas of Pakistan
4 Doing molecular work on thermocycler, gel appratus, 31 gel documentation system and nano drop
5 Disease incidence of bacterial canker on peach, apricot 34 and plum in district Attock in year 2015.
6 Disease incidence of bacterial canker on peach, apricot 34 and plum in district Attock in year 2016.
7 Disease incidence of bacterial canker on peach, apricot and 35 plum in district Rawalpindi in year 2015.
8 Disease incidence of bacterial canker on peach, apricot and 35 plum in district Rawalpindi in year 2016.
9 Disease incidence of bacterial canker on peach, apricot 36 and plum in district Khushab in year 2015.
10 Disease incidence of bacterial canker on peach, apricot 36 and plum in district Khushab in year 2016.
11 Disease incidence of bacterial canker on peach, apricot 38 and plum in district Abbottabad in year 2015.
12 Disease incidence of bacterial canker on peach, apricot and 38
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plum in district Abbottabad in year 2016.
13 Disease incidence of bacterial canker on peach, apricot 39 and plum in district Mansehra (Tehsil Mansehra) in year 2015.
14 Disease incidence of bacterial canker on peach, apricot 39 and plum in district Mansehra (Tehsil Mansehra) in year 2016.
15 Disease incidence of bacterial canker on peach, apricot 40 and plum in district Mansehra (Tehsil Oghi) in year 2015.
16 Disease incidence of bacterial canker on peach, apricot 40 and plum in district Mansehra (Tehsil Oghi) in year 2016.
17 Disease incidence of bacterial canker on peach, apricot and 41 plum in district Mansehra (Tehsil Balakot) in year 2015.
18 Disease incidence of bacterial canker on peach, apricot and 41 plum in district Mansehra (Tehsil Balakot) in year 2016.
19 Disease incidence of bacterial canker on peach, apricot and 44 plum in district Swat in year 2015.
20 Disease incidence of bacteria canker on peach, apricot and 44 plum in district Swat in year 2016.
21 Disease incidence of bacterial canker on peach, apricot 45 and plum in district Peshawar in year 2015.
22 Disease incidence of bacterial canker on peach, apricot 45 and plum in district Peshawar in year 2016.
23 Disease incidence of bacterial canker on peach, apricot and 46 plum in district Nowshera in year 2015.
24 Disease incidence of bacterial canker on peach, apricot and 46
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plum in district Nowshera in year 2016.
25 Peach Orchards with clear symptom of gummosis on 47 stem 26 Apricot fruits with bacterial canker symptoms collected 47 from field
27 Isolation of bacteria on nutrient agar media 53 28 Florescence of bacterial isolates under UV light 53 29 Formation of loop in KOH test 53 30 Disinfection of fruits with 50% ethanol 57 31 Inoculation of bacterium using pin prick method 57 32 Formation of canker symptoms after 4-5 days of incubation 57
33 Re-isolation of pathogen from developed symptoms 57 34 Syringomycin bioassay showing zone of inhibition 60 35 Inoculation of bacterial isolate into the leaf of tobacco plant 64
36 Hypersensitive response on tobacco plant 64 37 Phylogenetic tree constructed using maximum likelihood 74 method of 32 Pseudomonas syringae pv. syringae isolates from stone fruits using 16S rRNA primers
38 Phylogenetic tree constructed using maximum likelihood 75 method of 11 Pseudomonas syringae pv. morsprunorum isolates from stone fruits using 16S rRNA primers
39 Phylogenetic tree constructed using maximum likelihood 76 method of Pseudomonas syringae isolates from stone fruits using 16S rRNA primers with other isolates submitted in NCBI from the world
40 Phylogenetic tree of 8 Pss and 2 Psm isolates constructed 78 using gyrB primer
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41 Dendrogram of P. syringae pv. syringae isolates with 79 other isolates collected from NCBI data base using gyrB primers
42 Amplified PCR product with approximately 1453 bp 80 fragments using virD4 primers and Plasmid DNA was used as template
43 Amplified PCR product with approximately 513 bp 81 fragments using virB1 primers and Plasmid DNA was used as template
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List of Appendices
Appendix No. Page
1 Preparation of Nutrient Agar (NA) Medium 96
2 Preparation of Potato Dextrose Agar (PDA) 96 Medium
3 Preparation of King’s B (KB) Medium 96
4 Preparation of Levan Media 97
5 Preparation of Oxidase Solution 97
6 Preparation of Arginine dihydrolase Solution 97
7 Preparation of Gelatine Media 98
8 Preparation of Aesculin Media 98
9 Preparation of Tyrosinase Media 98
10 Preparation of L-Tartrate Media 99
11 Aligned Sequences obtained from NCBI 100
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List of Acronyms
µl Micro liter
DNA Deoxyribose Nucleic Acid
CFU Colony Forming Units cm Centimeter d Days
Fe Iron
Pss Pseudomonas syringae pv. syringae
Psm Pseudomonas syringae pv. morsprunorum g/gm Gram h Hour
L Liter
L. Linnaeus
NA Nutrient Agar lbs Pounds
M Molar min Minute ml/mL Milliliter mM Millimolar
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KB King’s B Media
NB Nutrient Broth oC Degree Celsius
P Phosphorus
PCR Polymerase Chain Reaction psi Per Square Inch
RNA Ribose Nucleic Acid rpm Revolution Per Minute
RT-qPCR Real Time Quantitative Polymerase Chain Reaction vol. Volume wt. Weight
DI Disease Incidence
MEGA Molecular Evolutionary Genetic Analysis
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ACKNOWLEDGEMENTS
Allah Almighty, the eternal of this universe. The most Beneficent, The
Merciful, The Gracious and The Compassionate whose bounteous blessings gave me potential, thoughts, talented teachers, helping friends and opportunity to make this humble effort and enabled me to pursue and perceive higher ideas of life.
All praises and respects to Holy Prophet Hazrat Muhammad (S.A.W.W) whose blessings and exaltations flourished my thoughts and thriven my modest all glory to almighty efforts in the form of this write-up.
The work presented in this manuscript was accomplished under the inspiring guidance, generous assistance and obligated supervision of Prof. Dr. M. Inam-ul-
Haq, Professor at Department of Plant Pathology. He had given the author guidance and advice with great patience. His criticism and suggestions had been of much value during the writing of thesis.
I deem it utmost pleasure in expressing my gratitude with the profound thanks to Prof. Dr. Tariq Mukhtar and Dr. Muhammad Ashfaq (Associate Professor),
Department of Plant Pathology and Dr. Zahid Akram, Associate Professor,
Department of Plant Breading and Genetics, for providing me with strategic command at every step. I extend deep emotions of appreciation and gratitude for their valuable guidance.
I am also obliged to HEC for granting me fund for International Research
Support Initiative Program (IRSIP). I would also like to express deep gratitude to
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Prof. Dr. Mark Gleason, Professor at Department of Plant Pathology and
Microbiology, Iowa State University, Ames, 50011, USA and his team for his valuable guidance and support.
I offer my profound obligations to my respectable father, my honorable mother, my brothers, sister and my family members for their encouragement, help and moral support throughout the study period.
Last but not the least; Friends (Amjad Shahzad Gondal, Sajjad Hyder,
Muhammad Saeed, Muhammad Farooq Aslam, Muhammad Shahid, Adnan Ahmad,
Muhammad Ibrahim Tahir, Muhammad Shahjahan, Sufyan Butt, Abdul Sattar, Nasir
Mehmood, Muhammad Fahim Abbas and Usama Latif) are the comrades of the battle, the battle to generate knowledge, sift myths and facts and to remove ambiguity. I express my thankful feelings for all my friends and colleagues.
Raees Ahmed
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ABSTRACT
Stone fruits are the major source of livelihood in some areas of Khyber-Pakhtunkhwa
(KPK) Province, Baluchistan Province and northern areas of Pakistan. According to the production data of Food and Agriculture Organization (FAO), 2014 Pakistan ranked 6th in apricot production all over the world, but the production of apricot and plum was decreased to thousands of tons from 2012 to 2014 (FAO, 2014). Several biotic and abiotic constrains involved in low production of peach, apricot and plum.
Regarding biotic factors bacterial diseases especially bacterial canker of stone fruits have major concern, it has been reported from almost all the countries producing stone fruits. However, in Pakistan no work has been done on pathological aspects of stone fruits. Depending on that scenario this study was proposed to determine the incidence and prevalence of bacterial canker disease in KPK and Punjab provinces of Pakistan. There was 100 % prevalence found in both the provinces of Pakistan and incidence of bacterial canker varies in different districts of KPK and Punjab but found that incidence was increased in every orchard in every location of Punjab and
KPK in 2nd year (2016). Highest disease incidence was in peach and apricot orchards of Murree area (Province Punjab) that was 66 and 54 % respectively in 2015.
However, the disease incidence increased to 71 and 56 % respectively in 2016. In plum orchards highest disease incidence was in Soan valley (54 %), it also increased
(57 %) in 2016. Similarly, in KPK province highest disease incidence in peach and apricot was in Swat i.e., 69% and 72% in 2015, next year it also increased to 75 and
67% respectively. In plum orchards highest disease incidence was in Nowshera that was 67% in 2015 and 71% in 2016. Pathogen was isolated from the infected samples
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(leaf, fruit and gum) and 64 isolates with gram negative having positive loop test results and also showed fluorescence under UV light were screened out as
Pseudomonas spp. Pathogenicity was done to check the pathogenic behavior of isolates and 43 isolates found pathogenic that develop sunken canker symptoms on fresh peach fruit and remaining isolates were discarded. During syringomycin bioassay isolates 32 isolates were found to be pathovar syringae that develop clear zone of inhibition from 1mm to 15mm. All pathogenic isolates were subject to biochemical characterization using LOPAT and GATTa scheme and L-lactate utilization test. Again results were similar 11 isolates showed confusing results.
Further molecular characterization was done to identify pathovars and races using universal (16S rRNA) primer and one house keeping gene (gyrB gene) primer.
Phylogenetic analysis was done using Molecular Evolutionary Genetics Analysis
(MEGA) version 7 and it was found that 32 isolates were P. syringae pv syringae and the remaining isolates were P. syringae pv. morsprunorum race 1. There was no pathovar morsprunorum race 2 isolate found from all the visited districts of KPK and Punjab provinces of Pakistan.
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Chapter 1
INTRODUCTION
Bacteria colonize on or inside plant organs on its host plants, of which roots and leaf are the best characterized organs for bacteria to colonize. Bacteria are classified to phyla, Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria associated with plants. Morphologically bacteria are microscopic unicellular with size range 1-2 µm and also invisible with naked eye, only visible under microscope. Bacterial communities associated with plants may be beneficial or some time not beneficial.
Epiphytic bacterial communities that are present on plant surfaces while endophytic bacterial communities live inside the plants and some of these bacteria are transients while others are residents. Among all the microbes in nature, on maturity bacteria are the one that colonize plants very successively. Some bacteria colonize the roots of plants and become beneficial for plants like rhizobium on leguminous crops, Nitrogen fixing bacteria in nodules while other groups of bacteria colonize plant roots to cause infectious diseases like root rot in chickpea etc.
Pathogenic bacteria are responsible to cause serious plant diseases all over the world (Vidhyasekaran, 2002) somehow fungi or viruses are also disease causing agents but damage due to these are less that cause economic losses less than bacteria
(Kennedy and Alcorn, 1980). Immunity/resistance is another factor in many plants to harbor pathogens asymptomatically that is without development of symptoms.
1.1 STONE FRUITS
Term stone fruits referred to “drupe” these are fruits having "stone" inside exocarp (Skin) and mesocarp (Flesh). This stone is made with hard endocarp and
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inside the stone there is a seed. Stone fruits include so many fruits like peach, plum, apricot, nectarines, lychees, walnuts, almonds and cherries. Stone fruits belong to the genus Prunus and family Rosacea. Stone fruits require severe winter to fulfill its chilling requirement to set flowering in coming cold spring and also require dry summer with warm temperature for complete ripening of fruits.
Stone fruits especially some peach varieties have 750 to 850 chill hours to set their flowers bit earlier and ultimately these varieties set fruits early in the season, so called early maturing crops. These varieties are suitable for areas having short but sever winter and not for the areas having longer cold because they set early flowering that may be damaged by frost injury. While those areas having longer sever winter varieties having 900 to 1,000 chill hours are suitable to grow because they can tolerate more cold during chilling period and they set their flowering late so they are late maturing varieties. From June to September mostly stone fruit crops become mature and delicious fruits must present in markets. Mostly stone fruits are susceptible to frost injury or low temperature injury because they are native to world’s warmer climates. Also frost in spring is very responsible to damage of blooms that appear in early spring on stone fruits.
Stone fruits are also perishable crops having very short shelf life, ultimately vulnerable to insects and diseases especially brown rot diseases in nectarines, plums and peaches. Stone fruits must be stored at 0 °C temperature with high humidity to increase its shelf life because they are fruits known as climacteric that must ripen continually after picking from the tree, so become more perishable having consumed in two weeks after harvesting.
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1.2 NUTRITION VALUE OF STONE FRUITS
Stone fruits are highly nutritive and delicious fruits rich with vitamins, fibers and minerals. Stone fruits are rich source of Vitamin A, B1, B2 and C, also contain minerals like Ca, P, Fe and K. Stone fruits also have some medicinal values like Peach fruit kernel is very good for blood circulation and also give relief in chronic constipation. Stone fruits have very low calories and sugar content also stone fruits have low fat contents that’s why they are very useful in weight management. Apricot, a stone fruit contains “vitamin A” equal to amount that is daily requirement for a person and 20 % “vitamin C” as it contains approx. 3.5 mg in a medium sized apricot.
Peach fruit is good for skin and also contain nutrients that are supportive for vision in human. Apricot fruit is also very good for heart and eyes because they are low in fat so good to manage cholesterol level. Apricots are also helpful for digestion if eaten before meal. Plum and cherry fruit is very good for skin protection because it contains antioxidants and is used in cosmetics, also plum and cherry fruit is a good source of fiber and potassium which are good for normal functioning of muscles. In short, healthy diet is not complete without stone fruits consumption.
1.3 STONE FRUITS IN WORLD
According to an estimate worldwide stone fruit production is more than 40 million tons annually and the approximate area for stone fruit production is 5 million hectares. Peach is considered as the third largest fruit specie with overall production is more than 18.42 million tons around the word (FAO, 2014).
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China is the major producer of peach, about 50% of total peach production in the world is produced by China every year but more than 1 million tons peach per year is also produced by USA, Italy and Spain. Average production in these countries may approach to 20 tons per hectare but 40 tons per hectares is also achieved by growers. Peaches and nectarines are important crops of USA, Greece, Australia and
South Africa that accounts more than 80% of their consumptions.
United Kingdom and Germany imports fresh plum in a large amount from
China, Spain, USA and South Africa. Japan also import mostly dried plum from
USA. Sweet cherry is mostly produced in Iran, Turkey and USA with an average production of 2.2 million tons. Similarly, Russia and Poland is major producer of sour cherries with average production of 1.3 million tons are largely produced in
Russia or in Eastern European countries such as Poland.
Turkey, Iran and Uzbekistan achieved 25-30 tons per hectare yield of apricot that is considered to be the best yield all over the world and overall 3.9 million tons apricot produced annually all over the world. According to US per capita, from last 10-20 years consumption of stone fruits in some countries become relatively static. As peach and nectarines consumption in USA is 2.7 kg per year and 0.6 kg per year for plums.
1.4 STONE FRUITS IN PAKISTAN
Some Stone fruits like peach, apricot, plum and cherries play a vital role in country’s economy and these are the major source of lively hood in Khyber
Pakhtunkhwa, Northern areas and Baluchistan. In Pakistan Khyber Pakhtunkhwa,
Northern areas and Baluchistan are best suitable for the production of stone fruits due to its environmental conditions best suitable for the production of peach, plum
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apricot and nuts. Total cropping acreage of apricot fruit in Khyber Pakhtunkhwa is
2.0 (000) ha with production of 14.0 (000) tons, peach is 5.6 (000) ha, with production of 30.8 (000) tons and plum fruit is 3.0 (000) ha, with production of 27.0
(000) tons (MinFAL, 2010-11).
Among stone fruits apricot ranked 1st in Pakistan according to its production and peaches are on 2nd with less production and then plum. In Pakistan mostly stone fruits are produced in district Attock, tehsil Murree in district Rawalpindi, district
Khushab, district Peshawer, Swat district, Mingora, Hazara, North Waziristan,
Chitral, some areas of district Quetta, district Pishin, district Loralai, most of the area of Qilla Saifullah and Qilla Abdullah, district Mastung, district Kalat and also in
Northern areas of Pakistan. According to FAO STAT, 2014 production of Apricot,
Peach and plum in Pakistan was 17,0504 tonns, 66,792 tonns and 54,304 tonns respectively with an average production area of 26,950 ha, 13,988 ha and 7,019 ha respectively.
In Pakistan so many peach varieties are grown i.e., early grand, florida king
6-A and 8-A are grown in district Peshawar and swat regions while golden early, shah pasand and shireen are major cultivars of peach grown in Balochistan. In case of Plum fazle mananai, faramusa beauty and late mananai are grown in stone fruit growing areas of Pakistan. As far as apricot is concerned, In Pakistan swat selection, red flesh early and old cap varieties are grown in stone fruit growing areas of
Pakistan.
Ahmed et al. (2008) reported that 10436 hectares of land is under fruit plantation but mostly plantation is irregularly grown orchards in crop fields or in yards.
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Also reported that in northern areas of Pakistan annual production of cheery was 1160 tons with 400 hectares of cultivation area. Northern areas of Pakistan are abundant with fruits that are scattered and fruit trees include mostly cherries, apple, peaches, plums and apricots etc. Environmental conditions of northern areas of Pakistan that contain very hard winder with comparatively cool spring followed by dry and hot summer is very suitable for the production of fruit trees especially stone fruits.
1.5 DISEASES OF STONE FRUITS
Although stone fruits are highly nutritive but the production of stone fruits not only in Pakistan, also all over the world was decreasing from last 3-4 years. There are so many factors involved in the decrease in production of stone fruits, biotic and abiotic. Among biotic factors diseases of stone fruits paly a very important role in the reduction of annual yield of stone fruits.
Ivanova, 2007 described that one of the largest pathological problem and factor that limit the successful cultivation of apricot in many countries is a very serious decease having causal organism P. syringae pv. syringae. Among stone fruits diseases anthracnose, bacterial canker, brown rot, peach leaf curl, plum pox, powdery mildew and peach scab are most common diseases all over the world. All of these diseases and pathogens are reported all over the world but in Pakistan no work was published on pathological aspects of stone fruits, its diseases and its pathogens.
According to Bhat et al., 2006 there are so many fruit trees in homes when surveyed northern areas, in average 70 different fruit trees present. Bhat et al., 2006 reported an extensive survey of two districts of Kashmir valley and identified that syringae and morsprunorum pathovars of P. syringae was responsible for bacterial canker disease
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of stone fruits. Also reported average disease incidence was 46.7% and 23.8% on twigs and scaffold limbs respectively and percentage disease intensity (PDI) was 22.5% on twigs as well as 9.3% on limbs in Baramulla district, similarly in Srinagar district, 38.9% and
23.3% of average disease incidence of bacterial canker was calculated on twigs and scaffold limbs respectively with 19.9% and 8.0%, PDI on twigs and limbs respectively.
According to Agrios (2005), P. syringae causes diseases of more than 180 different species of fruits, vegetables and ornamentals not only annual also of perennial crops.
1.6 BACTERIAL CANKER OF STONE FRUITS
Causal organism of bacterial canker is Pseudomonas syringae Van Hall. And on stone fruits two pathovars of P. syringae are responsible for disease development i.e., P. syringae pv. syringae and P. syringae pv. morsprunorum.
Bacterial canker disease of stone fruits affect almost all the parts of stone fruit trees but leaf, fruit and flower buds are more susceptible to disease. Among the symptoms of bacterial canker disease, blossom blast and twig die back in early summer is the clear symptom of the disease, lowers become brown and ultimately die. On leaves water soaked spots may appear which turn to brown with the age of leaves. At maturity canker drop out to give “shot hole” effect. On fruits clear symptom of bacterial canker is sunken spot having underlying gum with dark centers. Execution of gum from the bark is also a clear symptom of bacterial canker disease and after removing surface of bark from sunken areas, inside tissues are in orange to brown in color with sour smell.
As far as life cycle is concerned bacteria over winter in canker margins, surface of tress, in buds and in the grasses and shrubs of orchards. After severe
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winter, in wet spring bacteria ooze out from the margins of cankers and multiply on the flower buds. Bacteria spread by rain splashes and by wind. Bacteria enter into the plant via injuries and natural openings. Most favorable conditions for diseases are frequent rain periods with cool temperature and wind.
Table 1. Nutritional values of some stone fruits.
http:// www.eatingforenergy.com/ stone-fruit
Table 2. According to FAOSTAT, 2014 stone fruits production tons.
Pakistan Turkey India USA Serbia Australia China
Apricot 170504 278210 13999 58876 29655 10313 87921
Peach 66792 608513 260000 959983 91348 70349 12452377
Plum 54304 265490 225000 231800 401452 15777 6256906
Table 3. According to FAOSTAT, 2014 stone fruits production area in hectares.
Pakistan Turkey India USA Serbia Australia China
Apricot 26950 117907 5000 4379 5290 6250 21805
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Peach 13988 44070 37800 50602 8012 10067 728354
Plum 7019 20027 27600 27721 130000 3020 1829357
Figure 1. Map of Pakistan showing Khyber-Pakhtunkhwa, Punjab and Baluchistan
Provinces.
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Figure 2. Map showing districts of Province Baluchistan, Pakistan.
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Figure 3. Map showing districts of KPK Province and Northern areas of Pakistan.
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According to Vicente and Roberts, 2007 a survey was carried out in 2000-01 on wild cherry (Prunus avium) woodland plantations and nurseries and 20 out of the
24 plantations visited were found with bacterial canker symptoms. He reported that on the basis of serology, physiology and biochemically from wild cherry, sweet cherry and Prunus spp., 54, 22 and 13 isolates respectively were characterized and found that all the isolates were Pseudomonas syringae. Similarly, Cameron, 1962 and Mohammadi et al. (2010) also reported that bacterial canker is a serious problem in stone fruit growing areas all over the world.
According to Severin et al. (1986) and Hetherington (2005) all the organs of plant were susceptible to this disease with clear symptoms of canker or bud death as well as the execution of gum/wax from the injured portions of trunk are aboveground symptoms of bacterial canker. Symptoms like canker and necrosis present on branches of trees are important for the field identification of bacterial canker disease. According to Ogawa and English (1991) reduction of yield in stone fruits is directly due to canker development resulting tree decline and bud or flower death due to blast.
Some pioneer work was done and a survey of deciduous fruit diseases in northern Baluchistan was conducted (Haque et al., 1992). Later on in 1993, Haque et al. conducted a survey by the name “Some new diseases of fruit crops in Pakistan”.
In 1993, consultancy report on “Diseases of Horticultural Crops in Northern Areas” was published (Haque, 1993) and it was reported that the disease incidence from 40-
60% is present due to bacterial cankers.
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Keeping in mind the major constrains of stone fruit industry in Pakistan the objective of this study was developed to check the current status of bacterial canker disease in stone fruit growing areas of Khyber Pakhtunkhwa (KPK) and Punjab and to isolate its pathogen for further characterization using biochemical tests and molecular tools.
Objectives:
1. Determination of current status of bacterial canker on stone fruits growing
in Punjab and Khyber Pakhtunkhwa (KPK).
2. Biochemical characterization of the causal organism of the disease.
3. Molecular characterization of local isolates of the pathogen.
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Chapter 2
REVIEW OF LITERATURE
2.1 BACTERIAL CANKER OF STONE FRUITS
Bacterial canker disease referred as usual diseases name that are caused by
Pseudomonas syringae pv. syringae as their symptoms are almost same except bacterial dieback disease caused by P. syringae pv persicae (Bultreys and Kaluzna,
2010). According to (Bradbury, 1986; Young et al., 1996; Gardan et al., 1999) it’s a polyphagous plant pathogenic bacteria having 9 genomospecies and more than 50 pathovars can cause diseases on above 180 plant species of annual as well as perennial crops (Agrios, 2005).
Palleroni (2005) reported characteristics of P. syringae as they are Gram negative with a thin cell wall, have one or more flagella on its poles. These are aerobic, straight or with slight curve rods and also motile in nature that can cause diseases on stone fruits i.e., sweet cherry, plum, peach and apricot (Scortichini et al.,
2003; Vicente and Roberts, 2007; Fiori et al., 2003; Renick et al., 2008; Gilbert et al., 2009; Kaluzna et al., 2010b).
According to Kotan and Sahin, 2002 in three provinces of turkey about 80% trees of apricot fruit were infected with bacterial canker disease showing death of leaf and fruit buds and tree decline in apricot orchards. Dieback of peach branches in Izmir city of Turkey was observed with 10% disease incidence of bacterial canker
(Ozaktan et al., 2008).
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Scortichini (2006) also reported from Italy that in apricot orchards 30% of young apricot seedlings were infected with P. syringae pv. syringae show symptoms of dieback of twigs and tree decline. In Oregon state of USA bacterial canker was also reported in orchards of sweet cherry13 (S potts et al., 2010).
2.2 BIOCHEMICAL CHARACTERIZATION
P. syringae pv syringae strains can be identified traditionally by its physiology, nutritional and biochemical characteristics as well as its pathogenic behavior on peach seedlings (Little et al., 1998; Scortichini et al., 2003; Vicente and
Roberts 2003, 2007; Gilbert et al., 2009, 2010). According to Karimi-Kurdistani and
Harighi (2008) diseased samples were processed for isolation of bacteria using nutrient agar media to confirm 2-3mm diameter white, round and slight convex colonies of P. syringae pv. syringae. Also reported that these were stained and found gram negative, selective media King’s B was used and colonies show fluorescence under UV light. Species of Genus Pseudomonas can be easily differentiated using phenotypic character that include antibiotic productions such as syringomycin bioassay, shape of cells, position and presence of flagellum as well as growth of bacteria in different growth conditions (Peix et al., 2009).
Lelliott et al. (1996) reported that out of total fifteen biochemical tests, for the determination and differentiation of Pseudomonas spp. Only five tests with name
LOPAT are enough to determine species among pathogenic species of
Pseudomonas. LOPAT test can be used to differentiate species of phytopathogenic
Pseudomonas species (Bultreys and Kaluzna, 2010; Kaluzna et al., 2012). According
38
to Kaluzna et al. (2012) Lactate utilization and set of four tests named GATTa tests can be used to differentiate pathovars in plant pathogenic bacteria P. syringae.
In controlled conditions, several times studies have been carried out to check the freezing temperature against bacterial canker disease development on stone fruit trees (Cazorla et al., 1998). According to Süle and Seemüller (1987) disease severity of bacterial canker infection by P. syringae pv. syringae on stone fruit leaves inversely proportional to the freezing temperature i.e., with the increase of freezing temperature disease severity increases.
Kennelly et al., 2007 reported that P. syringae causing fruit tree canker have the ability to produce toxins like syringomycin that can produce pores by targeting plasma membrane of host, these toxins belong to the phytotoxin class that is cyclic lipodepsinonapeptide (Bender et al., 1999). Syringomycin toxin is responsible for the formation of necrotic tissue in host tree and plant signal molecules are involved for the expression of syringomycin gene especially present in the leaves of sweet cherry (Mo et al., 1991).
On the basis of biochemical characterization i. e., LOPAT and GATTa’s tests
27 isolates of bacteria isolated from diseased samples of apricot, peach, plum and sour, sweet cherry collected from Tehran Province of Iran were identified as P. syringae pv. syringae (Mohammadi et al., 2010). Vicente et al. (2004) also reported that on the basis of physiology and biochemical assay 54 isolates collected from wild cherry, 22 isolates from sweet cherry and from peach, plum and apricot 13 isolates were identified as P. syringae pv. syringae causing bacterial canker. Typical symptoms of bacterial canker were observed, also confirmed by pathogenicity test in apricot orchards in Turkey and identified as P. syringae pv. syringae (Kotan and Sahin, 2002).
39
2.3 MOLECULAR CHARACTERIZATION
To identify different pathovars of P. syringae isolates polymerase chain reaction (PCR) was used and declared to identify pant pathogens it become a very useful tool (Abu-Ashraf et al., 2000). Scortichini et al. (2003) done comparison between
101 isolates of P. syringae pv. syringae isolated from collected diseased samples or obtained from international culture collection bank using Rep-PCR with BOX primers.
To analyze the pathogens diversity of bacterial diseases of stone fruits Rep-PCR has been used as vey common and effective molecular method. Two races pf pathovars morsprunorum have homogeneity with each race and can be differentiated very rapidly using this method (Ménard et al., 2003; Vincente and Roberts, 2007).
Recently research using these methods to check the diversity between pathovars of P. syringae and also among two races of pathovar morsprunorum confirmed homogeneities in their DNA regions (Gilbert et al., 2009; Kaluzna et al.,
2010a). Ménard et al. (2003) reported that recently DNA hybridization was used to classify pathovars of P. syringae and reported that race 1 of P. syringae pv. morsprunorum belong to genomospecies 2 and race 2 belong to genomospecies 3 whereas pathovar syringae belong to genomospecies 1.
Multilocus Sequence Typing (MLST) used to analyze housekeeping genes and PCR amplification was used to amplify internal transcribed spacer using 16S and 23S rRNA primers for these genes to determine discrimination between isolates of P. syringae (Sarkar and Guttman, 2004; Hwang et al., 2005; Kaluzna et al.,
2010b). Karimi-Kurdistani and Harighi (2008) detected vir B1 and vir D4 genes using polymerase chain reaction (PCR) and amplified expected 1453bp and 513bp
40
product using D4 and B1 primers respectively using 93.1kb plasmid as DNA template.
41
Chapter 3
MATERIALS AND METHODS
A systemic survey of stone fruit growing areas of Punjab (that include
Rawalpindi, Islamabad and Attock) and KPK (that include Abbottabad, Mansehra,
Peshawar, Nowshera, Mingora and Swat) was done to compute the incidence and prevalence of bacterial canker disease in Peach, Plum and Apricot. Stone fruit orchards were visited in May 2015 and again in May 2016 next year, when fruits were on the ripening stage to collect the data and samples for the confirmation of disease. Diseased samples (leaf, stem, gum and fruit) were collected during survey.
3.1 COLLECTION OF SAMPLES
Samples were collected during each survey from each location two times in one growing season and were tagged by placing in a paper bag for isolation of pathogen. All the bags were tied and labeled. During survey leaf, gum, bud and fruit samples were collected from all the orchard having average 8-9 years old peach, plum and apricot trees.
3.2 DETERMINATION OF DISEASE INCIDENCE AND DISEASE
PREVALENCE
The diseased plant (that were under the attack of bacterial canker, showing disease symptoms) was noted for the determination of disease incidence. Number of locations in a district varies according to the intensity of stone fruit orchards in that area. In an orchard, total 15 plants were selected using X-manner. Out of these
15 plants, number of diseased plants were determined. After that the incidence of
17
42
disease was computed by the formula:
No. of infected trees Disease Incidence (%) = × 100 Total No. of trees selected
Number of locations in each area were surveyed according to the intensity of stone fruit tree/orchards.
Prevalence was calculated by using this formula:
No. of Locations Showing Bacterial Canker Prevelence %age = × 100 Total No. of Locations Visited
3.3 ISOLATION AND PURIFICATION OF PATHOGENIC BACTERIA
FROM DISEASES SAMPLES
From each location diseased samples (leaf, bud, stem, gum and fruit) were collected and were taken for isolation. Isolation was done from canker and stem lesions of stone fruit trees. Isolation of pathogen was also done from canker lesions on fruits and gum samples collected during survey. Sterilization of surface tissues was done with sodium hypochlorite followed by washing with sterile distilled water. Maceration of excised small pieces with canker margins in sterile distilled water and steaking of resulting suspensions on nutrient agar (NA) media was done followed by incubation at 27 ± 2 0C. Then after 2-3 days, single colonies were sub-cultured on selective iron deficient media i.e., King’s Broth medium (KB) (King and Raney, 1954).
3.4 INITIAL SCREENING OF BACTERIAL CULTURES
Initial screening was done using gram staining, Loop test and UV light for the confirmation of Pseudomonas spp.
3.4.1 Gram Staining
43
Fixation of bacteria was done on the slide with low flame heating. Then
44
crystal violet solution spreaded on the smear. Then washing was done after 30 seconds with tap water followed by spreading iodine on the slide for 1 minute again washing was done. After washing decolorization was done using 95% ethanol for 30 seconds. Then safranin was used for counter staining and again washing was done. After drying slides were observed under microscope
(Schaad, 1988).
3.4.2 KOH (Loop) Test
One drop of potassium hydroxide solution was placed on a glass slide. One day cultures of bacterial isolates were removed aseptically from plates using wire loop and were placed on the solution. Bacterial cultures were stirred for 20 seconds and were observed for the formation of loop (Suslow et al., 1982).
3.4.3 Florescent under UV Light
Bacterial cultures were grown on KB media and incubation was done at 27
±2 ºC for 1-2 days. Bacterial strakes were then observed under UV light for florescence (Kaluzna et al., 2012).
3.5 PRESERVATION OF CULTURE
For preservation of isolates two methods were used, preservation in glycerol at -20 0C and preservation in mineral oil at room temperature.
3.5.1 Preservation in Glycerol
For preservation in glycerol, purified cultures were grown on King’s B media. Then fully grown cultures were scraped from the surface of media and were
45
placed in 1-2ml screw capped self-standing tubes having 50 % neutral glycerol.
These culture tubes were stored at -20 0C.
3.5.2 Preservation in Mineral Oil
Agar slants were used for the preservation of stock cultures in oil. After the sterilization process agar slants were prepared by placing them on slightly inclined position. Mineral oil was also sterilized using hot air at 170 0C for an hour. When growth of bacteria was observed on the slants, sterilized mineral oil was added on about 1 cm above the tip of slants and were screw capped to store at room temperature.
3.6 PATHOGENICITY TESTS
Inoculums of bacterial isolates were prepared from 24-48h old cultures of
P. syringae isolates grown on king’s B media. Fresh peach fruits were collected from market and 50% ethanol was used for dipping of peach fruit to disinfect the fruit followed by washing with SDW three times to remove the residues of ethanol from fruit and drying was done using tissue paper. Peach fruits were put on filter paper soaked in water in the petri plates. Three peach fruits were used per bacterial isolate for inoculation and control was inoculated with distilled water. Inoculation of each peach fruit in three places upto 2mm depth with bacterial suspension was done using sterile needle. After inoculation incubation was done on 22 ± 2 0C for
3-4 days. After each 24 h of incubation development of symptoms were observed on inoculated fruits.
3.7 SYRINGOMYCIN BIOASSAY
46
Indicator fungus (Geotrichum candidum) for syringomycin bioassay was obtained from 1st Fungal Culture Bank of Pakistan, University of the Punjab,
Pakistan. Suspension of fungus was prepared in sterile distilled water. For syringomysine bioassay 24-48 h old cultures of P. syringae pvs. were used. Potato
Dextrose Agar medium (PDA) was used to streak P. syringae isolates and were incubated at 27 ± 2 0C for 2-3 days. When growth of bacteria appeared on artificial media (PDA), G. candidum inoculum was sprayed on whole plates. Again plates were placed in incubator at 27 ± 2 0C for 1-2 days and after incubation zone of inhibition for the growth of fungus was observed.
3.8 BIOCHEMICAL CHARACTERIZATION OF BACTERIAL
CANKER PATHOGENS
For the growth of P. syringae pv. syringae and morspnorum 25-28 0C temperature is optimum. According to Lelliott and Stead (1987) LOPAT tests can be used for the determination of species in Pseudomonas genera that was also tested by
Kaluzna et al., 2012. For species determination following five tests referred as
LOPAT was used.
3.8.1 Levan Production Test
For levan test petri plates were prepared with nutrient agar media having additional % sucrose. These plates were streaked and were incubated at 27± 2 °C for
1-2 days. Colony characteristics were observed after each 24 hours to compare the results with Lelliott and Stead (1987).
3.8.2 Oxidase Test
47
Wurster's blue reagent (N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride solution provided by Sigma) was prepared (1% w/v) with sterile distill water and sterilized filter paper was soaked in the prepared solution. Bacterial isolates grown on KB media were transfer to filter paper using sterilized tip.
Immediate change in colour was compared with Lelliott and Stead (1987) LOPAT scheme.
3.8.3 Potato Soft Rot Test
Fresh potato tubers were used for this test. Potato tubers were sliced and sterilized, then placed in a sterilized petri plate having moist filter paper. Then bacterial culture grown on KB media after 24-48 h was stab-inoculated on the center of potato slices. Results were observed after incubation for 1-2 days at 27 ± 2 °C.
3.8.4 Arginine Dihydrolase Test
For arginine dihydrolase test a semi-liquid media was prepared using 0.01% peptone, 0.5% NaCl, K2HPO4, phenol red, L-arginine and agar and was poured in test tubes. Then 1-2 days old culture of bacterial isolate from KB media was transferred in the tubes containing semi-liquid media and was covered with mineral oil. Then after 2-4 days of incubation change in colour of media was observed. Tubes not covered with mineral oil was set as control.
3.8.5 Hypersensitivity Test
Tobacco plants were maintained in Glass house to check the hypersensitivity reaction on tobacco plants 1-2 days old bacterial cultures were used to prepare a dense suspension having 108 cells/ml. Application of thick
48
suspension was done in the mesophyll of tobacco leaf. After 24 h of inoculation symptoms were observed.
After specie determination in genus Pseudomonas, P. syringae have more than
50 pathovars according to its host. To distinguish different pathovars in P. syringae
Lelliot and Stead (1987) reported GATTa tests that are also used by Kaluzna et al. (2012) for distinguishing pathovars along with L-lactate utilization test. Following five tests to distinguish pathovars in P. syringae isolates were also used in this study.
3.8.6 Gelatine Hydrolysis Test
For gelatine test a solid media was prepared using 0.3% yeast extract,
0.5% peptone and 12% gelatin and poured in test tubes. Then 24h old culture of bacterial isolates already cultured on KB media was transferred to the tubes having solid gelatine media and incubated for 7-10 days at 18 °C for the confirmation of results.
3.8.7 Aesculin Hydrolysis Test
For aesculine test 1% peptone, 0.1% esculine, 0.05% ferric citrate and 2% agar was used to prepare a semi solid media for inoculation of bacterial isolates.
Then inoculation of 1-2 days old culture of bacterial isolates was done followed by again incubation of 1-2 days at 27 ± 2 °C to change the color of medium to black.
3.8.8 Tyrosinase Activity
Test tubes were prepared with semi solid medium for the inoculation of bacterial isolates using sucrose, casamino acid, L-tyrosine, KH2PO4, MgSO4.7H2O
49
and agar. 1-2 days old cultures were used for inoculation and after incubation of 7-
10 days change in color of medium was observed.
3.8.9 Tartrate Utilization Test
For tartrate utilization test liquid suspension was prepared by using
NH4H2PO4, 0.2% KCl, 0.02% MgSO4.7H2O, 4% alcohol bromothymol blue solution
(1 ml), 7.0 pH was maintained and was transferred to test tubes wit screw caps.
Inoculation of 1-2 days old bacterial cultures was done followed by incubation. The change in color of medium was observed.
3.8.10 L-lactate Utilization Test
Again a semi solid medium was prepared by using 0.1% (NH4)3PO4, KCl,
MgSO4, 0.004% bromothymol blue (alcoholic solution), agar and 1% lactate and was poured into the test tubes. Inoculation of 24-48 h was done on prepared medium and change in color of medium was observed after incubation.
3.9 MOLECULAR CHARACTERIZATION AND GENETIC
VARIABILITY
3.9.1 Primers Used in Study
For the amplification of PCR product already published primers (Table 4) were used for the amplification of 16 s RNA (Moore et al., 1996), vir D4, vir B1 gene (Karimi-Kurdistani and Harighi, 2008) and gyrB gene (Yamamoto et al., 2000).
3.9.2 Extraction of Genomic DNA
50
Extraction of Total genomic DNA was done using 24 h old bacterial cultures grown on LB broth Media using Gene Jet DNA Purification Kit K#0721 provided by Thermo Scientific following all the instruction of manufacturer.
1. Bacterial cells grown on LB broth overnight in a shaking incubator were
harvested in a 1.5 ml microcentrifuge tube using 10 min centrifugation at
5000 x g. After centrifugation the supernatant was discarded.
2. Digestion was done by using 180 μl into the tube containing bacterial pellet.
Then after adding 20 μl Proteinase K solution made the suspension uniform
by vortexing or pipetting.
3. Digested samples were incubated at 56 °C in water bath for 30 min with
51
vortexing after every 5 min.
4. After incubation 20 μl RNase A solution was added and again incubation was
done at room temperature for 10min after mixing the mixture with vortexing.
5. Then 200 μl Lysis Solution was added with vortexing to made homogeneous
solution.
6. 400 μL ethanol 50% was added and pipetting was done to mix the solution.
7. Then lysate solution was transferred to the column with collection tube
provided by the manufacturer and at 6000 x g column was centrifuged for 1
min. After centrifugation collection tube was discarded and column was
placed in a new collection tube.
8. 500 μl Wash Buffer I was added to the column and 1 min centrifugation was
done at 8000x g. Flow through was discarded.
9. Then again washing was done by adding 500 μl Wash Buffer II and again
centrifugation was done at 13000 x g for 3 min.
10. Collection tube was discarded after centrifugation containing flow through
and column was placed into a new sterile microcentrifuge tube.
11. 50-200 μl Elution Buffer was added to the center of column then incubation
was done at room temperature for 2 min and centrifuged at 8000 x g for 1 min.
12. Column was discarded and purified DNA was stored at -20 °C in a deep freezer.
3.9.3 Plasmid Extraction
52
Plasmid extraction was done using 24 h old bacterial cultures grown on LB broth Media using Gene Jet Plasmid Mini Prep Kit K#0503 provided by Thermo
53
Scientific following all the instruction of manufacturer.
1. Bacterial cells were harvested by 2 min centrifugation at 6800 x g at room
temperature and supernatant was decanted.
2. Then 250 μl Resuspension Solution was added to the pellet collected and
mixed by vortexing.
3. Lysis was done by adding 250 μl Lysis Solution and mixed the solution by
inverting the tube 3-4 times.
4. Then neutralization was done by adding 350 μl Neutralization solution and
mixed by inverting and then centrifuged at 8000 x g for 5 mins.
5. Then solution was transferred to the column and collection tube provided by
manufacturer and centrifugation was done for 1 min.
6. Washing was done by adding 500 μl Wash Solution and centrifugation was
done for I min. This step was repeated 2 times and then collection tube was
discarded.
7. To elute plasmid 20- 50 μl elution buffer already provided was used by
placing to the center of column and was stand for 2 min at room temperature
followed by centrifugation for 2 min at 8000 x g.
8. Purified product was stored at -20 oC in a refrigerator.
3.9.4 PCR Amplification
3.9.4.1 PCR for 16s rRNA
54
Genomic DNA was used as template DNA to amplify targeted 16s rRNA genes using polymerase chain reaction (PCR) with T100 Thermo Cycler (Bio-Rad
Germany). Forward primer 27F having 8-27 position according to E. coli numbering and reverse primer 1492 R having 1492 to 1507. 50 µl reaction mixture contains 25
µL GoTaq® Green Master Mix (Promega), 1 µl of each forward and reverse primers,
2 µL of template DNA, 1 µl Taq DNA polymerase and remaining volume 20 µl was maintained with DEPEC treated water. The conditions for 30 cycles of PCR contains initial denaturation for 2.5 mins at 94 ºC followed by denaturation for 1 min at another 94 ºC, annealing of primer for 1 min at 52 ºC and at 72 ºC extension for 2 min. Elongation of reaction was done with 10 min final incubation at 72 ºC (Karlson et al., 1993).
3.9.4.2 PCR for virB1 and virD4 genes
For the amplification of virB1 and virD4 genes, extracted plasmid was used as
DNA template in PCR reaction. And reaction mixture contains 50 μl rection having
25 μl GoTaq® Green Master Mix (Promega), 1 μl of each forward and reverse primer
(virB1 and virD4), 1 μL DNA polymerase, 0.5 μl Plasmid DNA as template and 20.5
μl DEPEC treated water to made final volume of reaction. And reaction conditions were maintained as according to the Karimi-Kurdistani and Harighi (2008) as one cycle of initial denaturation for 3 min at 94 °C for 4 min followed by 29 cycles of denaturation for 1min at 94 °C, primer annealing for 1 min at 56 °C for virB1 or 58 °C for virD4 and extension for 1 min at 72 °C. Elongation of reaction mixture with one cycle of final extension for 7 min at 72 °C (Karimi-Kurdistani and Harighi, 2008).
55
3.9.4.3 PCR for gyrB gene
Amplification of gyrB gene was done using reaction mixture and conditions reported by Yamamoto et al. (2000). PCR reaction mixture contains total volume of
100 µL having 50 µl GoTaq® Green Master Mix (Promega), 2.5 µl of each primer
(forward and reverse), 2 µl DNA polymerase enzyme and 2 µl genomic DNA as template and 43.5 µl DEPEC treated water to made ultimate volume. PCR reaction conditions followed were one cycle of initially denaturation for 5 min at 94 ºC and then 30 cycles of denaturation, primer annealing and extension at 94 ºC for 1min, 60
ºC for 1min and 72 ºC for 2 min respectively. Then elongation with incubation at 72
ºC for another 10 min was done (Yamamoto et al., 2000).
3.9.5 PCR Purification
PCR purification was done using Gene Jet PCR Purification Kit K#0702 following manufacturer instructions.
1. Equal ratio in volume of Binding Buffer was dissolved in PCR reaction
mixture and was mixed by vortexing.
2. Solution from step 1 was transferred to the purification column provide by
the manufacturer and centrifugation for 1 min was done at 8000 xg. Followed
by discarded the flow through.
3. Washing was done by adding 700 μl washing buffer into the purification
column and again centrifuge for 1min at 8000 xg was done. Again flow
through was discarded and column was again centrifuged to remove residues
completely.
56
4. After transferring the column into new collection tube PCR product was
eluted by adding 20-50 μl elution into the center of column followed by
centrifugation at maximum speed.
5. Column discarded after collected purified DNA product in 1.5 ml
microcentrifuge tube.
3.9.6 DNA Clean and Concentration
TM To obtain optimum DNA concentration DNA Clean & Concentrator -5 was used provided by Zymo Research using standard protocol provided by Manufacturer.
1. DNA binding buffer was added in a ratio of 2:1, 2 volumes of buffer to 1
volume DNA in a microcentrifuge tube and mixed the solution with
vortexing.
2. Mixture was then transferred into the column with collection tube provided
by the manufacturer.
3. After 30 seconds of centrifugation column was removed and flow through
was discarded.
4. Then added 200 μL DNA wash buffer for washing followed by centrifugation
at 5000 x g for 30 sec. This step was repeated 2 times.
After adding ≥6 μL DNA Elution Buffer, 1 min incubation was done at room temperature. And collected concentrated DNA was stored at -20 oC.
3.9.7 Quantification of Purified PCR Product
NanoDrop™ 2000c Spectrophotometers was used to quantify the purified
PCR product. NanoDrop sample loading unit was first blanked by loading aliquot of
57
blank onto the loading unit and then click on blank to measure the blank measurement. After that 1 μl of each sample was loaded one by one to measure the quantity of DNA present in the purified PCR products.
3.9.8 Sequencing and Phylogenetic Analysis
16s RNA and gyrB gene sequence obtained for isolates were compared with already submitted sequences available in NCBI data base. Manipulation of obtained forward and reverse sequences were done with BioEdit program and sequence alignments were done using Clustal W program (Thompson et al., 1997). MEGA version 7.0 was used for the phylogenetic analysis (Tamura et al., 2013) using the
Maximum likely-hood method.
58
Table 4: Primer sequences used for PCR analysis (Karimi-Kurdistani and Harighi,
2008; Moore et al., 1996; Yamamoto et al., 2000).
59
Figure 4. Doing molecular work on Thermocycler, Gel Apparatus, Gel
Documentation System and NanoDrop.
Chapter 4
RESULTS AND DISCUSSION
4.1 SURVEYS FOR DISEASE INCIDENCE
Extensive visit of peach, plum and apricot orchards of stone fruit growing areas of Punjab and KPK were done in the year 2015 and 2016. In each growing season stone fruit orchards were surveyed to check the disease incidence, prevalence and collection of diseased samples for isolation, purification and identification. Survey was done in X-manner and 15 trees in each orchard were selected. During survey it was noted that early grand, florida king 6-A, 8-A, golden early and shireen were the most commonly grown varieties of peach on overall basis in the country. Indian Blood was also grown in some of the areas as late variety. Similarly, swat selection, red flesh early and old cap were mostly grown varieties of apricot where as in some areas nari and travet were also grown. Fazle mananai, faramusa beauty, late mananai, grand Duke,
Ruby red and Red beauty were the varieties of plum grown mostly in Pakistan.
4.2 DISEASE INCIDENCE AND DISEASE PREVALENCE OF
BACTERIAL CANKER OF STONE FRUITS
Disease incidence and prevalence of Stone fruits (Peach, Apricot and Plum) in Punjab province i.e., Districts Attock, District Rawalpindi (Including Islamabad),
District Khushab was determined by visited stone fruit orchards first time in 2015 and then again all the orchards were again visited in 2016.
60
4.2.1 Punjab Province
In 2015, three locations were visited in district Attock. It was noted that in all these sites, the disease prevalence of bacterial canker of stone fruit was almost
100%. Maximum disease incidence of bacterial canker on peach and apricot was 32 noted in Madrotha that was 67% and 60% respectively, while on Plum (27%) in
Hazron was recorded. The lowest value of disease incidence on Peach, Apricot and
Plum was in Fateh jang (40%), Hazron (33%) and Madrotha (13%) respectively in
2015 (Figure 5).
Same orchards in all the locations were again visited in the next year (2016) and it was calculated that there was increase in disease incidence. In case of peach and apricot fruit disease incidence was 80% and 67% respectively calculated from Madrotha in 2016 while in case of plum disease incidence calculated in 2016 was increased to 40% in
Hazron. (Figure 6). Overall comparison of two growing seasons clearly indicate the increase in diseases incidence in all the locations visited in district Attock.
In district Rawalpindi, stone fruits were only grown in Murree tehsil, because environmental condition in Murree are suitable for stone fruit production. In 2015, five locations in tehsil Murree were visited and disease incidence of bacterial canker on peach and apricot fruit was 60% in Bassian that was increased in 2016 up to 73% in case of peach in Bassian while 67% in case of apricot in Mussiari. While disease incidence was 67% in plum orchards of Numble when calculated in 2015 that was also increased to 80% when calculated in 2016. (Figures 7-8).
In district Khushab of Punjab province stone fruits orchards were only in soon valley. During survey five locations in 2015 maximum disease incidence
61
was 53% in peach orchards calculated from Kotli. In apricot orchards, highest disease incidence was calculated from Anga and Sakesar villages that was 53%, while in plum orchards 67% disease incidence was highest in Makrumi village of district Khushab.
62
100
80
60
40
20 % Disease Incidence Disease %
0 Hazron Fateh jang Madrotha Peach Apricot Plum
Figure 5. Disease incidence of bacterial canker on peach, apricot and plum in district
Attock in year 2015. Percentage Disease incidence on Y-axis and locations
on X-axis. Disease incidence is the mean that was calculated from selected
15 trees in each orchard and 3 orchards in each location.
100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Hazron Fatheh jang Madrotha Peach Apricot Plum
Figure 6. Disease incidence of bacterial canker on peach, apricot and plum in district
Attock in year 2016. Percentage Disease incidence on Y-axis and locations
on X-axis. Disease incidence is the mean that was calculated from selected
15 trees in each orchard and 3 orchards in each location.
63
100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Ausia Dewal Mussiari Bassian Numble Peach Apricot Plum
Figure 7. Disease incidence of bacterial canker on peach, apricot and plum in district
Rawalpindi in year 2015. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated from
selected 15 trees in each orchard and 3 orchards in each location.
90 80 70 60 50 40 30
20 % Disease Incidence Disease % 10 0 Ausia Dewal Mussiari Bassian Numble Peach Apricot Plum
Figure 8. Disease incidence of bacterial canker on peach, apricot and plum in district
Rawalpindi in year 2016. Percentage Disease incidence on Y-axis and
64
locations on X-axis. Disease incidence is the mean that was calculated from
selected 15 trees in each orchard and 3 orchards in each location.
70 60 50 40 30 20
% Disease Incidence Disease % 10 0 Uchalli Anga Kotli Makrumi Sakesar Peach Apricot Plum
Figure 9. Disease incidence of bacterial canker on peach, apricot and plum in district
Khushab in year 2015. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated from
selected 15 trees in each orchard and 3 orchards in each location.
90 80 70 60 50 40 30 20 % Disease Incidence Disease % 10 0 Uchalli Anga Kotli Makrumi Sakesar Peach Apricot Plum
Figure 10. Disease incidence of bacterial canker on peach, apricot and plum in
district Khushab in year 2016. Percentage Disease incidence on Y-axis
65
and locations on X-axis and locations on X-axis. Disease incidence is the mean that was calculated from selected 15 trees in each orchard and 3 orchards in each location.
66
In 2016, noted that disease incidence in district Khushab was increased that was 67% in peach, 67% in apricot and 80% in plum from Kotli, and Sakesar. (Figures
9-10). In 2016 disease incidence was increased in all the three districts and in all the orchards in every location visited this was an alarming situation for researchers and breeders as well as the stone fruit producers of Pakistan.
4.2.2 KPK Province
In KPK province of Pakistan, five locations in district Abbottabad were surveyed and in 2015 it was calculated that maximum disease incidence was 73% in peach orchards of Bangnotar and sjikote, also 73% in apricot orchards of Goreeni.
While it was 53% in plum orchards of Birote and Nambal in 2015 (Figure 11), again in 2016 the disease incidence was increased to 80% in peach orchards of Bangnotar and 80% in apricot orchards of Goreeni while 67% in plum orchards of Nambal in district Abbottabad (Figure 12).
In district Mansehra three tehsils were visited i.e., Tehsil Mansehra, Oghi and
Balakot. In 2015 nine locations were visited in Tehsil Mansehra and highest disease incidence was 60% in peach orchards of Hilkot and Jaborri while 73% DI was calculated from apricot orchards of Jaborri (Figure 13). In case of plum in 2015 highest DI was 67% in Jaloo. The DI was increased to 73% in peach and apricot orchards of Jaborri while 73% in plum orchards of Jaloo (Figure 14).
Similarly, in tehsil Oghi six locations were visited and in 2015 highest DI was 60%, 53% and 60% in peach, apricot and plum orchards of Darband, Dalborri and Shergarh respectively (Figure 15) that was again increased to 67% in peach
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orchards of Darband, 60% in apricot orchards of Darband and Shamghara, while 67 in plum orchards of Shergarh in tehsil Oghi (Figure 16).
100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Bangnotar Birote Nambal Sajikote Goreeni Peach Apricot Plum
Figure 11. Disease incidence of bacterial canker on peach, apricot and plum in district
Abbottabad in year 2015. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
90 80 70 60 50 40 30
20 % Disease Incidence Disease % 10 0 Bangnotar Birote Nambal Sajikote Goreeni Peach Apricot Plum
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Figure 12. Disease incidence of bacterial canker on peach, apricot and plum in district
Abbottabad in year 2016. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
100 80 60 40 20
0 % Disease Incidence Disease %
Peach Apricot Plum
Figure 13. Disease incidence of bacterial canker on peach, apricot and plum in
district Mansehra (Tehsil Mansehra) in year 2015. Percentage Disease
incidence on Y-axis and locations on X-axis. Disease incidence is the
mean that was calculated from selected 15 trees in each orchard and 3
orchards in each location.
100 80 60 40 20
0 % Disease Incidence Disease %
Peach Apricot Plum
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Figure 14. Disease incidence of bacterial canker on peach, apricot and plum in
district Mansehra (Tehsil Mansehra) in year 2016. Percentage Disease
incidence on Y-axis and locations on X-axis. Disease incidence is the
mean that was calculated from selected 15 trees in each orchard and 3
orchards in each location.
100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Darband Nika Pani Shergarh Dalborri Shamdhara Karori Peach Apricot Plum
Figure 15. Disease incidence of bacterial canker on peach, apricot and plum in district
Mansehra (Tehsil Oghi) in year 2015. Percentage Disease incidence on Y-axis
and locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
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80 70 60 50 40 30
20 % Disease Incidence Disease % 10 0 Darband Nika Pani Shergarh Dalborri Shamdhara Karori Peach Apricot Plum
Figure 16. Disease incidence of bacterial canker on peach, apricot and plum in district
Mansehra (Tehsil Oghi) in year 2016. Percentage Disease incidence on Y-axis
and locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
100 80 60 40 20
% Disease Incidence Disease % 0 Garhi Hangrai Kewal Mohandri Talhat Garlat Habib Ullah Peach Apricot Plum
Figure 17. Disease incidence of bacterial canker on peach, apricot and plum in
district Mansehra (Tehsil Balakot) in year 2015. Percentage Disease
incidence on Y-axis and locations on X-axis. Disease incidence is the
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mean that was calculated from selected 15 trees in each orchard and 3
orchards in each location.
100 80 60 40 20 0 % Disease Incidence Disease % Garhi Hangrai Kewal Mohandri Talhat Garlat Habib Ullah Peach Apricot Plum
Figure 18. Disease incidence of bacterial canker on peach, apricot and plum in
district Mansehra (Tehsil Balakot) in year 2016. Percentage Disease
incidence on Y-axis and locations on X-axis. Disease incidence is the
mean that was calculated from selected 15 trees in each orchard and 3
orchards in each location.
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Six locations in tehsil Balakot were also visited for the calculation DI.
In 2015, 60% highest DI was calculated from peach orchards of Garlat and also
60% DI was calculated in apricot orchards of Kewal and Garlat villages while
67% DI was calculated from plum orchards of Garlat village also (Figure 17).
In 2016, DI was again increased to 73% in Garlat in case of peach and 67% in apricot orchards of Hangrai and Garlat villages while 73% in plum orchards of
Talhat village (Figure 18).
District Sawat is the main hub of stone fruit production in KPK province of
Pakistan. Ten locations of stone fruit producing areas of district swat were surveyed in 2015 and 2016 to calculate the disease incidence in district sawat. In 2015, highest
DI was 73% in peach orchards of Kota and Drush Khela, while 67% DI was calculated in apricot orchards of Thana and Kota. In case of plum orchards highest
DI was calculated 60% tha was in Thana and Drush Khela in district sawat. (Figure
19). In next year DI was increased in every location and it was calculated that DI was increased to 80% in peach orchards of Matta in 2016 while 73% in apricot orchards of Kota and Matta. Highest DI of 67% was calculated in plum orchards of
Nawakaley (Figure 20). Overall highest disease incidence was recorded in Swat district that was 47-80% in peach orchards, 40-73% in apricot orchards and 40-67% in plum orchrads. Disease prevalence was recorded100% in Swat district.
Among five locations surveyed in Peshawar district in 2015, maximum disease incidence of bacterial canker was recorded 73% on peach orchards in Urmar Payan
(Figure 21) which was gradually increased in 2016 up to 80% (Figure 22). Highest disease incidence (60%) of bacterial canker was recorded on apricot orchards in
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Tarnab while on plum orchards disease incidence (60%) was recorded in Urmar Payan and Lala Kaley in 2015 (Figure 21) which was increased to 73% in apricot orchards of Tarnab in 2016 and also 73% in plum orchards of Lala Kaley in 2016 (Figure 22).
In district Nowshera three areas were visited and maximum disease incidence was found in Akbar Pura on peach which was recorded 60% in year
2015 (Figure 23) which was increased to 67% in year 2016 (Figure 23). In case of apricot 60% disease incidence was recorded in Wazir Garhi (Figure 23) which was increased to 73% in next year (Figure 24). While in case of plum orchards, in 2015 highest disease incidence was calculated in Akbar Pura that was 60% and was also increase to 67 % in 2016 (Figure 24). In Punjab and KPK,
100% disease prevalence of bacterial canker of stone fruits was calculated in all the visited districts of Punjab and KPK. Results reveled that bacterial canker disease was prevailed in all the orchards surveyed during 2015 and 2016.
The results of current study were very similar with the previous findings of Yildiz et al. (2016) that in three provinces (Adana, Mersin and Hatay) of
Turkey, total 68 orchards of stone fruits were surveyed and about 5-30% disease incidence was recorded in plum and apricot orchards during 2014. Similar studies were also done by Spotts et al. (1990) that orchards of sweet cherry were evaluated in 1982 to 1986 annually for bacterial canker. Pathogen was isolated from margins of canker symptoms and was noted that P. syringae pathovars cause bacterial canker on 13% trees in 1982 while increased to 25% in 1986. Death rate was 17% in infected trees with P. syringae and also high disease incidence was observed in trees near old orchards. The findings of current study were similar
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with previous findings that bacterial canker infected trees increased in production year 2016 similar with findings of Spotts et al., 1990.
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100 90 80 70 60 50 40 30 20
% Disease Incidence Disease % 10 0
Peach Apricot Plum
Figure 19. Disease incidence of bacterial canker on peach, apricot and plum in
district Swat in year 2015. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
100 80 60 40 20
% Disease Disease % Incidence 0
Peach Apricot Plum
Figure 20. Disease incidence of bacterial canker on peach, apricot and plum in
district Swat in year 2016. Percentage Disease incidence on Y-axis and
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locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
100
80
60
40
% Disease Incidence Disease % 20
0 Musazai Surazai Urmar Payan Tarnab Lala Kaley Peach Apricot Plum
Figure 21. Disease incidence of bacterial canker on peach, apricot and plum in district
Peshawar in year 2015. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated from
selected 15 trees in each orchard and 3 orchards in each location.
100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Musazai Surazai Urmar Payan Tarnab Lala Kaley Peach Apricot Plum
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Figure 22. Disease incidence of bacterial canker on peach, apricot and plum in district
Peshawar in year 2016. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Akbar Pura Qasim Ali Baig Wazir Garhi Peach Apricot Plum
Figure 23. Disease incidence of bacterial canker on peach, apricot and plum in district
Nowshera in year 2015. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
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100 90 80 70 60 50 40 30
% Disease Incidence Disease % 20 10 0 Peach Apricot Plum Akbar Pura Qasim Ali Baig Wazir Garhi
Figure 24. Disease incidence of bacterial canker on peach, apricot and plum in district
Nowshera in year 2016. Percentage Disease incidence on Y-axis and
locations on X-axis. Disease incidence is the mean that was calculated
from selected 15 trees in each orchard and 3 orchards in each location.
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Figure 25. Peach Orchards with clear symptom of gummosis on stem.
Figure 26. Apricot fruits with bacterial canker symptoms collected from Mangora.
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According to Kennelly et al. (2007) that bacterial canker caused by P. syringae pathovars is one of devastating disease of stone fruit orchards worldwide and cause significant losses. Observed symptoms were closely resembled with the findings of Kennelly et al. (2007) that during blooming period clear blast on blooms and dieback as well as in mid-season lesions on leaves and later on lesions on fruits with gummosis on stem and also on fruits were observed.
4.3 ISOLATION AND PURIFICATION
From all the districts of Punjab province and KPK province in 2015, total of
800 composite samples were collected that include leaf samples with clear canker margins, gum samples on the stem and fruit samples having canker symptoms.
Similarly, in next year 2016 all the orchards in all the district were again surveyed and total of 800 leaf, gum and fruit samples were again collected for the isolation of pathogen associated with the diseases. Collection of 10 leaf, 5 fruit and 5 gum samples were taken from all sides of a selected tree to made a composite sample.
After placing infected samples on nutrient agar media and incubation different colonies were developed on the media after 2 -3days. From these bacterial growths expected growths of P. syringe that were translucent growth on nutrient agar media (Kaluzna et al., 2012) were picked with sterilized needle and were re-streaked onto KB media plates to obtain the pure culture of Pseudomonas spp.
4.4 INITIAL SCREENING OF BACTERIAL ISOLATES
All the recovered isolates on KB media were then initially screened out on the basis of gram test, loop test and UV light. From all the recovered isolates,
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64 isolates were gram negative and fluorescent under UV light, these bacterial isolates were again cultured on KB media to multiply the isolates for the preparation of bacterial suspension for pathogenic studies. As previous findings shown that P. syringae is a phytopathogenic gram negative bacterial species and florescent under UV light which having more than fifty pathovars and nine genomospecies (Bradbury, 1986; Young et al., 1996; Gardan et al., 1999). In previous studies it was reported that it was responsible to cause diseases in cultivated cherry, plum, peach and apricot as well as in wild cherry known to be very economically important in stone fruits producing countries of the world
(Scortichini et al., 2003; Vicente and Roberts, 2007; Renick et al., 2008; Gilbert et al., 2009; Kaluzna et al., 2010).
Initial screening results were according to the previous results of characterization of bacterial isolates on the basis of phenotypic features that include, Gram staining and isolate sensitivity to potassium hydroxide as described by Suslow et al., 1982 that all the
Pseudomonas spp. were Gram negative and positive for KOH sensitivity. Similarly, according to Hildebrand et al., 1988 Pseudomonas spp. produce fluorescent pigments when grown on KB media and were visualized under UV light. On the basis of these previous findings 64 isolates were gram negative, and positive in loop formation when subjected to KOH test (Figure 23C) and also showed florescence under UV light (Figure
23B) when cultured on KB media and were considered as Pseudomonas spp. (Table 5).
The results were also similar with the findings of Kaluzna et al., 2012 that describes morphology of bacterium as gram negative and florescent under UV light confirms
Pseudomonas spp. the current results were also similar with the findings of Kersters et
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al. (1996) that P. syringae bacterium belongs to Pseudomonas genus that include in subclass γ of Proteobacteria are gram negative when stained.
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Table 5. Screened isolates with their labeled name, their host and isolation source.
S. Isolation Gram Loop UV S. Isolation Gram Loop UV Isolates Host Isolates Host No. Source Test Test light No. Source Test Test light 1 PS-1 Peach Fruit - + + 20 PS-20 Peach Leaf - + +
2 PS-2 Peach Fruit - + + 21 PS-21 Peach Leaf - + +
3 PS-3 Peach Fruit - + + 22 PS-22 Peach Leaf - + +
4 PS-4 Peach Fruit - + + 23 PS-23 Peach Gum - + +
5 PS-5 Peach Fruit - + + 24 PS-24 Peach Gum - + +
6 PS-6 Peach Fruit - + + 25 PS-25 Peach Gum - + +
7 PS-7 Peach Fruit - + + 26 PS-26 Apricot Fruit - + +
8 PS-8 Peach Fruit - + + 27 PS-27 Apricot Fruit - + +
9 PS-9 Peach Fruit - + + 28 PS-28 Apricot Fruit - + +
10 PS-10 Peach Fruit - + + 29 PS-29 Apricot Fruit - + +
11 PS-11 Peach Fruit - + + 30 PS-30 Apricot Fruit - + +
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12 PS-12 Peach Fruit - + + 31 PS-31 Apricot Fruit - + +
13 PS-13 Peach Leaf - + + 32 PS-32 Apricot Fruit - + +
14 PS-14 Peach Leaf - + + 33 PS-33 Apricot Fruit - + +
15 PS-15 Peach Leaf - + + 34 PS-34 Apricot Fruit - + +
16 PS-16 Peach Leaf - + + 35 PS-35 Apricot Leaf - + +
17 PS-17 Peach Leaf - + + 36 PS-36 Apricot Leaf - + +
18 PS-18 Peach Leaf - + + 37 PS-37 Apricot Leaf - + +
19 PS-19 Peach Leaf - + + 38 PS-38 Apricot Leaf - + +
39 PS-39 Apricot Leaf - + + 52 PS-52 Peach Fruit - + +
40 PS-40 Apricot Leaf - + + 53 PS-53 Peach Fruit - + +
41 PS-41 Apricot Gum - + + 54 PS-54 Peach Fruit - + +
42 PS-42 Apricot Gum - + + 55 PS-55 Peach Fruit - + +
43 PS-43 Plum Leaf - + + 56 PS-56 Peach Fruit - + +
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44 PS-44 Plum Leaf - + + 57 PS-57 Peach Fruit - + +
45 PS-45 Plum Leaf - + + 58 PS-58 Peach Fruit - + +
46 PS-46 Plum Leaf - + + 59 PS-59 Peach Fruit - + +
47 PS-47 Plum Leaf - + + 60 PS-60 Peach Fruit - + +
48 PS-48 Plum Leaf - + + 61 PS-61 Peach Leaf - + +
49 PS-49 Peach Fruit - + + 62 PS-62 Peach Fruit - + +
50 PS-50 Peach Fruit - + + 63 PS-63 Peach Fruit - + +
51 PS-51 Peach Fruit - + + 64 PS-64 Peach Fruit - + +
- Represents gram negative bacterium isolate, + represents positive response PS in table stands for Pseudomonas syringae
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Figure 27. Isolation of bacteria on Figure 28. Fluorescence of bacterial
nutrient agar media. isolates under UV light.
Figure 29. Formation of loop in KOH test.
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4.5 PATHOGENICITY TEST
Bacterial suspension of all 64 isolates were made for pathogenicity trail and all bacterial isolates were tested for pathogenicity on fresh and healthy peach fruit
(Figures 30-32). Out of total 64 pure isolates 43 isolates were positive showing clear canker necrotic symptoms on peach fruits (Table 6) after incubation of 3-4 days at
27 ± 2 ºC. The results of pathogenicity were reconfirmed by re-isolating the bacteria from the symptoms developed on the peach fruits and same colonies of bacterial growth were recovered from the area of inoculation which confirms Koch’s postulates that these bacteria are pathogenic and develop same symptoms.
Bacterial isolates were considered as positive (+) which develop canker symptoms that were sunken spots with dark centers and also yellow hallow appeared around spots
(Figure 32) and those which failed to develop canker symptoms on peach fruits after incubation of 4 days were considered as non-pathogenic and were discarded. Forty-three positive samples were then further used for further studies. Pathogenicity test was one of common phenotypic diagnostic method for bacterial canker pathogen, that was conducted on different host plants as well as plant organs to confirm the pathogen (Vicente et al.,
2004; Ménard et al., 2003; Renick et al., 2008; Scortichini et al., 2003).
Our studies correspond with Kaluzna and Sobiczewski, 2009 that fruitlets of cherry can be used for pathogenicity test not only to check its virulence by canker development but also its pathovar differentiation was done. Similar results also showed from our studies that fresh peach fruits were also used to determine the
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virulence of bacterial canker pathogen and also its pathogenic and non-pathogenic isolates were isolated on the basis of symptom development.
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Table 6. Pathogenicity of bacterial isolates on fresh peach fruits using pin prick method.
S. No. Isolates Response S. No. Isolates Response S. No. Isolates Response
1 PS-1 - 17 PS-17 + 33 PS-33 + 2 PS-2 + 18 PS-18 - 34 PS-34 + 3 PS-3 + 19 PS-19 + 35 PS-35 - 4 PS-4 - 20 PS-20 + 36 PS-36 - 5 PS-5 - 21 PS-21 + 37 PS-37 + 6 PS-6 + 22 PS-22 + 38 PS-38 + 7 PS-7 + 23 PS-23 + 39 PS-39 + 8 PS-8 + 24 PS-24 + 40 PS-40 + 9 PS-9 + 25 PS-25 + 41 PS-41 + 10 PS-10 + 26 PS-26 - 42 PS-42 - 11 PS-11 + 27 PS-27 + 43 PS-43 - 12 PS-12 - 28 PS-28 + 44 PS-44 + 13 PS-13 + 29 PS-29 + 45 PS-45 + 14 PS-14 + 30 PS-30 - 46 PS-46 + 15 PS-15 + 31 PS-31 + 47 PS-47 + 16 PS-16 - 32 PS-32 + 48 PS-48 -
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S. No. Isolates Response S. No. Isolates Response S. No. Isolates Response
49 PS-49 + 55 PS-55 + 61 PS-61 +
50 PS-50 - 56 PS-56 + 62 PS-62 +
51 PS-51 + 57 PS-57 - 63 PS-63 -
52 PS-52 - 58 PS-58 - 64 PS-64 -
53 PS-53 + 59 PS-59 -
54 PS-54 + 60 PS-60 -
+ represented positive result as bacterial isolates were pathogenic develop bacterial canker symptoms
- represented negative result as bacterial isolates were non-pathogenic (No. symptom development)
Isolates highlighted as bold were discarded because they were non-pathogenic.
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Figure 30. Disinfection of fruits with 50% Figure 31. Inoculation of bacterium using
ethanol. pin prick method.
Figure 32. Formation of canker symptoms Figure 33. Re-isolation of pathogen from
after 4-5 days of incubation. developed symptoms.
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4.6 SYRINGOMYCIN BIOASSAY
Previous 43 isolates were further studied for the presence of syringomycin gene to check the isolates were belonged to pathovar syringae. After consecutive two incubation of 5-6 days at 27 ± 2 ºC, first after bacterial inoculation and 2nd after spray of indicator fungi on PDA plates clear zone of inhibition was observed which shows that bacteria having syringomycin gene can produce syringomycin toxin that can inhibit the growth of G. candidum.
Isolates showed zone of inhibition confirmed that these isolates have syringomycin gene that produced toxin to inhibit the growth of fungus clearly indicates that they belonged to the pathovar syringae of P. syringae bacterium
(Figure 34). But the remaining isolates were either belonging to P. syrinage pv. morsprunorum race 1 and race 2 which were also reported from stone fruit producing countries that can cause bacterial leaf spot on stone fruits. The studies should have pointed out that isolates have syringomycin gene. From total 43 pathogenic isolates
32 isolates produce syringomycin toxin which was clearly indicated by the result of growth inhibition of indicator fungus G. candidum grown on PDA. Some isolates inhibit growth of fungus resulting 13-15 mm zone of inhibition, some isolates developed less zone of inhibition and 11 isolates (Ps-2, Ps-8, Ps-15, Ps-17, Ps-22,
Ps-24, Ps-33, Ps-39, Ps-40, Ps-45 and Ps-55) not developed any zone of inhibition
(Table 7). Therefore, all the isolates were further studied and identified by using biochemical tests to confirm the pathovars of P. syringae.
Results of syringomycin bioassay in current study was similar with the previous findings of Gašić et al., 2012 reported that strain KFB 0103 of P. syringae
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pv. syringae produced syringomycin toxin resulting in producing zone of inhibition when G. candidum was applied as indicator fungus in syringomycin bioassay.
Results were also confirmed with the similar results of Bultreys and Gheysen, 1999 that “genomospecies” syringae was evaluated for syringomycin production using G. candidum strain MUCL 31566 on PDA to inhibit the growth of fungus. Similar studies were also reported by Hu et al., 1998 used Aspergillus niger as indicator fungus for biological assay.
4.7 BIOCHEMICAL CHARACTERIZATION OF PATHOGENIC
ISOLATES
All the isolates shown positive levan formation test when inoculated on sucrose supplemented nutrient agar media (Table 8) develop mucoid, dome like colonies on sucrose media. Also reaction with oxidase, rottening of potato slice and
Arginine dihydolysis was negative that means violet colour on filter paper was not appeared when isolates were applied on filter paper soaked with tetramethyl-p- phenylenediamine dihydrochloride solution. They were not able to produce pectolytic ability so potato slices were not rotten when isolates were applied and incubated (Table 8) that confirms all the isolates were P. syringae. The results were similar with the previous findings of El-Siesy (2007), he reported that three isolates shown positively with hypersensitivity test, levan formation while negative in case of potato soft rot and oxidase activity were characterized as P. syringae isolates.
Also the findings were very close to the previous reports that to identify the specie level of Pseudomonas set of five tests named LOPAT test are very useful tool that proves positive results for Levan production and hypersensitive response
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(Figures 35-36) while negative for oxidase test and arginine test but variable results with soft rot of potato (Kaluzna et al., 2012).
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Figure 34. Syringomycin bioassay showing zone of inhibition.
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Table 7. Syringomycin bioassay of pathogenic bacterial isolates.
Inhibition Inhibition Isolates Response Isolates Response zone (mm) zone (mm) PS-2 - 0 PS-31 + 8 PS-3 + 11 PS-32 + 11 PS-6 + 12 PS-33 - 0 PS-7 + 9 PS-34 + 12 PS-8 - 0 PS-37 + 14 PS-9 + 9 PS-38 + 11 PS-10 + 11 PS-39 - 0 PS-11 + 13 PS-40 - 0 PS-13 + 15 PS-41 + 15 PS-14 + 13 PS-44 + 9 PS-15 - 0 PS-45 - 0 PS-17 - 0 PS-46 + 8 PS-19 + 11 PS-47 + 11 PS-20 + 7 PS-49 + 12 PS-21 + 12 PS-51 + 15 PS-22 - 0 PS-53 + 12
PS-23 + 9 PS-54 + 11 PS-24 - 0 PS-55 - 0 PS-25 + 15 PS-56 + 7 PS-27 + 14 PS-61 + 9 PS-28 + 7 PS-62 + 11 PS-29 + 9 + sign showing clear zone of inhibition having syringomycin gene
- sign shown fungal growth with no zone of inhibition that were not P. syringae pv.
syringae
Isolates highlighted in bold represented that they were not P. syringae pv. syringae.
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According to Lelliott and Stead (1987) results were similar with the current findings that P. syringae isolates have round and slightly white colonies, fluorescent and gram negative that were also positive for levan and hypersensitivity test while negatively responded for oxidase and soft rot on potato.
P. syringae have more than 50 pathovars on the basis of their hosts, two pathovars syringae and morsprunorum of P. syringae bacterium are responsible to cause bacterial canker disease on stone fruits. After LOPAT tests it was confirmed that 43 recovered isolates were P. syringae and further more confirmation of pathovars was done using GATTa and L-lactate utilization test was done developed by Lelliot and Stead, 1987. GATTa scheme confirmed that 32 isolates from total recovered 43 isolates were P. syringae pv. syringae shown positive Gelatin and aesculin hydrolysis while negative tyrosinase activity and tartrate utilization.
Remaining 11 isolates were totally opposite in case of GATTa tests. Ps-2,
Ps-8, Ps-15, Ps-17, Ps-22, Ps-24, Ps-33, Ps-39, Ps-40, Ps-45 and Ps-55 were negative in case of Gelatin and aesculin hydrolysis while positive in case of tyrosinase activity and tartrate utilization were confusing may be P. syringae pv. morsprunorum race 1 or race 2 (Table 9). Similarly, in case of L-lactate utilization test, isolates confirmed as P. syringae pv. syringae showed positive result by changing the color of media from green to blue after 2-3 days of incubation. 32 isolates showed positive while remaining 11 isolates showed negative results for L-lactate utilization test (Table 9).
According to Schaad (2001) it was reported that GATTa scheme was negative for Gelatin and aesculin hydrolysis while positive for tyrosinase and tartrate
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utilization and also negative for L-lactate utilization test, that was (--++-) in case of pathovar morsprunorum race 1 while in case of morsprunorum race 2 it may me positive or negative. This statement was further confirmed by the similar results of previously findings that pathovar morsprunorum race 2 was positive and sometimes negative when GATTa scheme and L-lactate utilization test was applied (Vicente and Roberts, 2007; Ménard et al., 2003; Gilbert et al., 2009).
Results of current study were also confirmed by comparing the previous findings of Kaluzna et al., 2012 who reported that different detection methods were used including LOPAT, GATTa, L-lactate utilization, pathogenicity and syringomycin bioassay for distinguishing species and pathovars under a project framework of COST 873 for bacterial pathogens of nuts and stone fruits.
Similarly, Kaluzna et al., 2012 also used GATTa scheme as biochemical characterization to confirm different pathovars of P. syringae. From total recovered 43 isolates 32 isolates were belonged to pathovar syringae of P. syringae bacterium but results of remaining 11 isolates were confused either these were pathovar morsprunorum race 1 and race 2 and also now a day’s biochemical characterization is not a reliable source for the confirmation of bacteria on advance levels. So to remove any chance of error all the 43 isolates were subjected to genetic studies for further confirmation of their pathovars and races associated with these pathovars.
For molecular confirmation multigene phylogeny was used using 4 sets of primers, one universal primer for bacterial identification and one house keeping
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gene i.e; gyrB gene, while other two genes were amplified using plasmid as template in PCR reaction.
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Figure 35. Inoculation of bacterial isolate into the leaf of tobacco plant.
Figure 36. Hypersensitive response on tobacco plant.
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Table 8. LOPAT scheme for the identification of P. syringae according to Lelliott and Stead (1987).
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103
104
Table 9. GATTa and Lactate test for the confirmation of pathovars in P. syringae according to Lelliott and Stead (1987).
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106
107
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4.8 MOLECULAR CHARACTERIZATION
4.8.1 16S rRNA Gene
On the basis of PCR results with universal primer (16S rRNA) sixteen isolates were selected having approximate 1500bp band size on 1% agarose gel using
1kb DNA ladder. PCR product of these sixteen isolates were purified and were send to Macrogen Korea for sequencing. Approximately 1400bp sequence was obtained with forward and reverse primers (Universal 16S RNA i.e., 27F and 1492R) and then all the sequences were aligned using Bio-Edit software and then final sequences were submitted to NCBI GenBank database.
MEGA 7.0 was used for phylogenetic analysis of all the sequences obtained with
16s RNA. Phylogenetic analysis revealed that the 32 isolates were 97-100% similar with the previously submitted isolates of P. syringae pv. syringae (Figure 37). Tree was constructed by using maximum likelihood method in MEGA version 7 and on the basis of genetic similarity tree was divided into two clusters. In Cluster-I: Ps-27, Ps-31 and
Ps-32 were closely resembled with each other while Cluster-II: 29 isolates were further divided into sister clads on the basic of their genetic similarity with each other. Cluster
II was divided into 8 sister clads and were genetically similar with P. syringae pv. syringe (KP753381) isolate of South Korea and isolate of china (KF500097).
The results were similar to the previous studies that amplification of 16s rRNA gene with PCR and then sequencing determines 97% or more similarity while comparison of 21 Pseudomonas species with already published strain of P. aeruginosa (Toschka et al., 1988).
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Similarly, remaining 11 isolates were blasted and were found genetically similar with P. syringae pv. morsprunorum and again maximum likelihood method was used to construct the phylogenetic tree and also closely related with the isolates of New Zealand (KF019104) and Korea (GU997635). Tree was divide into two sister clads and in Clad-I: Ps-45, Ps-55, Ps-2, Ps-8, Ps-15 and Ps-17 were genetically close to each other while in Clad-II: Ps-33, Ps-40, Ps-24, Ps-22 and Ps-39 were genetically close to each other (Figure 38).
All the isolates were then used in phylogenetic analysis with other isolates already reported in NCBI data base using maximum likelihood method and phylogenetic tree was constructed and it was found that all the isolates showed maximum similarity with isolates from New Zealand, Iran and Italy (Figure 39). Tree was constructed with 80% cut value to condense tree accurately and it was found that all the isolates were divided into 10 sister clads according to their similarity percentage and are closely related with isolates of New Zealand, and also with isolates of Italy and Iran.
4.8.2 GyrB Gene
All recovered isolates were subjected to PCR amplification and sequencing from
Macrogen Korea and DNA facility of Iowa State University and the sequences were blast and aligned. It was observed all the isolates were divided into clusters when analyzed using molecular phylogenetic analysis by maximum likelihood method. In cluster I, it was further divided into two sister clads. In first sister clad Ps-27 (MF377480), Ps-34
(MF377482) and Ps-37 (MF377487) characterized as pathovar syringe of P. syringae and
Ps-17 (MF401398) and Ps-34 (MF401399) was characterized as pathovar morsprunorum
110
of P. syringae. While in second cluster Ps-41 (MF377483), Ps-29 (MF377481), Ps-46
(MF377485), Ps-44 (MF377484) and Ps-47 (MF377486) were characterized as P. syringae pv. syringae (Fig. 40). All the isolates have 99 to 100% similarity with other P.
111
syringae pv. syringae and pv. morsprunorum isolates submitted in NCBI data base.
Similarly, when isolates of P. syringae pv. syringae was analyzed using molecular phylogenetic analysis by maximum likelihood method with isolates of other countries submitted in NCBI data base it was observed that tree was divided in to two clusters, in cluster one three isolates Ps-27, Ps-34 and Ps-37 were closely related with isolates of Japan, Iran and Spain. While other five isolates (Ps-29, Ps-
46, Ps-41, Ps-44 and Ps-47) were closely related with isolate of Australia in second cluster (Figure 41).
The results were compared with previous studies of Yamamoto et al. (2000) that thirty-one Pseudomonas species having 125 strains were subjected to phylogenetic analysis and two intrageneric clusters were observed i.e., IGC I and
IGC II. Similarly, according to Verhille et al., 1999 that when compared phylogenetic relationship of 16s rRNA with those of phylogenetic analysis of gyrB and rpoD sequences these were different with each other. A tree obtained from molecular phylogenetic analysis by maximum likelihood method showed that all the isolates were closely related with previously reported two genomospecies 1 and 3 of
P. syringae (Berge et al., 2014; Gardan et al., 1999; Sarkar and Guttman, 2004).
4.8.3 virB1 and virD4 Gene
Plasmid was extracted and then plasmid was visualized by running plasmid on 2% gel agarose and visualized under UV transluminator. It was noted that almost all the isolates had up to three plasmids, one having approximately
371 kb, second have 93.1 kb and third one have molecular weight 16.5 kb. Then
112
PCR was done using all three plasmids and also genomic DNA as templet in PCR studies using B1 and D4 primers to amplify virB1 and virD4 gene.
I
I
-
uster
Cl
I
-
uster
Cl
Figure 37. Phylogenetic tree constructed using maximum likelihood method of 32
Pseudomonas syringae pv. syringae isolates from stone fruits using 16S
rRNA primers.
113
I
-
Clad
I
I
-
Clad
Figure 38. Phylogenetic tree constructed using maximum likelihood method of 11
Pseudomonas syringae pv. morsprunorum isolates from stone fruits
using 16S rRNA primers.
114
Figure 39. Phylogenetic tree constructed using maximum likelihood method of
Pseudomonas syringae isolates from stone fruits using 16S rRNA
primers with other isolates submitted in NCBI from the world.
115
It was noted that expected 513 and 1453bp fragments of amplified DNA with primers B1 and D4 (Figures 42-43) was obtained when 93.1 kb molecular weight plasmid was used as template DNA in PCR reaction.
The results of current study were found close to the previous findings of Zhao et al. (2005) reported that strains of P. syringae having pPT23A plasmid family have one or more plasmids and using two sets of primers B1 and D4 amplified 513bp and
1453bp PCR fragment when 93.1 kb plasmid was used as template.
Also according to sundin et al. (2004) virB1 and virD4 genes were present in a complete sequence of pPSRI. Cao and Saier (2001) reported that virB1 and virD4 genes have great role in conjugation process because these genes are responsible in formation of such proteins that are involved in secretion system especially type 4
(T4SS) that is major secretion system in gram negative bacteria.
So depending upon the reports of previous findings, our study confirms the presence of virB1 and virD4 genes when 93.1 kb plasmid was used as templet and expected fragments were amplified successfully. Karimi-Kurdistani and Harighi
(2008) also reported that 93.1kb plasmid was extracted and was used as templet to amplify virD4 and virB1 genes to confirm P. syringae pv. syringae. Based on our results it confirms that P. syringae pv. syringae isolates had one to three plasmids, having one plasmid with 93.1 kb molecular weight was used to confirm presence of virB1 and virD4 genes on it.
116
Cluster I
Cluster II
Figure 40. Phylogenetic tree of 8 Pss and 2 Psm isolates constructed using gyrB
primer. Pseudomonas fluorescence as out group in phylogenetic analysis.
117
Cluster I
Cluster II
Figure 41. Dendrogram of P. syringae pv. syringae isolates with other isolates
collected from NCBI data base using gyrB primers.
118
Figure 42. Amplified PCR product with approximately 1453 bp fragments using
virD4 primers and Plasmid DNA was used as template.
119
Figure 43. Amplified band size of approximately 513 bp fragments using virB1
primers and Plasmid DNA was used as template.
120
SUMMARY
Bacterial canker is economically very important disease of stone fruit that is responsible for major yield losses in the major stone fruit producing countries of the world. This disease is caused by P. syringae pathovars and was reported from stone fruit producing countries of the world. However, in Pakistan there was no work done on disease of stone fruits. The current study was first time carried out on bacterial canker disease of stone fruits. The aim of this study was to investigate the current status of bacterial canker in stone fruit producing areas of Punjab and KPK also confirmation of pathovars and races of pathogen associated with the disease using biochemical and molecular tools. Incidence and prevalence of bacterial canker disease was calculated after two-year extensive survey of Punjab (Attock,
Rawalpindi and Khushab) and KPK (Peshawar, Nowshera, Abbottabad, Mansehra and Swat) in 2015 and 2016. 100 % disease prevalence was found in all the visited districts of Punjab and KPK. Disease incidence varies in all the visited districts but highest disease incidence in peach and apricot orchards in Punjab was in Murree that was 66 and 54 % in 2015 while it was increased to 71 and 56 % in 2016 respectively.
While in plum orchards, highest disease incidence was in Soan valley (54 %), it also increased (57 %) in 2016. Similarly, in KPK province highest disease incidence in peach and apricot was in Swat i.e., 69% and 72% in 2015, next year it was also increased to 75 and 67% respectively. In plum orchards highest disease incidence was in Nowshera that was 67% in 2015 and 71% in 2016. Infected samples (leaf, fruit and gum) were collected and isolation of pathogen was done using nutrient agar media purified on King’s B media. Initial screening was done on the basis of Gram staining, loop test and UV light. From all the recovered isolates 64 isolates were
82 121
screened out as Pseudomonas spp. and were test for pathogenicity on fresh peach fruits using pin prick method of inoculation. After pathogenicity test on fresh peach fruits 43 isolates were screened out as pathogenic and rest were discarded.
Syringomycin bioassay was done to confirm the pathovar syringae using Geotricum candidum. Clear zone of inhibition confirmed 32 isolates were pathovar syringae and rest were pathovar morsprunorum. For specie confirmation LOPAT scheme was followed and on the basis of results all the isolates showed positive levan and tobacco hypersensitive response while negative oxidase test, pectolytic activity on potato tubers and arginine dihydrolase test thus confirming all the 43 isolates were
Pseudomonas syringae. After specie confirmation pathovar was confirmed following GATTa scheme and L-lactate utilization test. It was observed that 32 isolates showed positive gelatine, aesculin hydrolysis and lactate utilization test while negative tyrosinase and tartrate tests confirming 32 isolates were
Pseudomonas syringae pv. syringae while other 11 isolates showed opposite response i.e., negative gelatine, aesculin hydrolysis and lactate utilization test and according to GATTa scheme remaining 11 isolates were belonged to pathovar morsprunorum either race 1 or race 2.
Molecular confirmation of isolates was done using multigene phylogeny using two sets of primers (16s rRNA and gyrB gene) while virB1 and virD4 gene primers were also used to amplify expected bands. DNA was extracted followed by
PCR amplification. Results were visualized with Gel documentation system on 1% agarose gel and were purified, concentration of PCR amplicons were measured with
Nano drop and were sent to DNA facility ISU for sequences. Obtained sequences were aligned and phylogenetic analysis with maximum likelihood method was done
122
using MEGA version 7.0. Results of BLAST and Phylogenetic analysis of isolates showed 99-100% similarity with already reported isolates of two pathovars of P. syringae (syringae and morsprunorum race 1) using universal primer (16s rRNA) and a house keeping gene primer (gyrB gene). There was no evidence found for P. syringae pv morsprunorum race 2 from any location visited.
123
CONCLUSIONS
This is the first report of bacterial canker disease on stone fruits from
Pakistan.
100 % prevalence was found in visited districts of Punjab and KPK.
Disease incidence was increased in all the visited districts of Punjab and KPK
in 2016.
Bacterial canker in Pakistan is mainly caused by Pss and Psm race 1.
There was no evidence found for Psm race 2 in stone fruit growing areas of
Punjab and KPK.
Multi gene sequences of Pakistani bacterial canker isolates of stone fruits are
now available in NCBI public database.
85 124
FUTURE DIRECTIONS
As bacterial canker found widely prevalent in Punjab and KPK so, warrants
strict quarantine and control strategies.
Mostly pathovar syringae found in Punjab and KPK so management
strategies should be taken accordingly.
Pathovar morsprunorum race 1 was found only in Swat district and is more
destructive that’s why incidence was highest in swat district, so control
strategies shout be adopted to prevent its spread.
There is need to develop resistant germplasm against two pathovars of
bacterial canker.
The results will be very useful for understanding the disease importance and
management strategies using different control measures.
The main cause of high disease incidence was pathogen overwintering on
stone fruit trees and fallen fruits in the orchards so proper sanitation measures
should be adopted to reduce the inoculum potential.
86 125
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APPENDICES
Appendix 1: Preparation of Nutrient Agar (NA) Medium
Recipe of Nutrient agar media was.
Ingredient Quantity Nutrient Agar Powder 28 g Distilled water 1000 ml Prepared nutrient agar powder provided by LabM was added to 1000 ml distilled water and was mixed on hotplate. Sterilization of media was done by autoclaving media at 121 ºC and 15 Psi pressure.
Appendix 2: Preparation of Potato Dextrose Agar (PDA) Medium
Ingredient Quantity Potato Dextrose Agar Powder 39 g Distilled water 1000 ml Prepared PDA powder provided by Phyto Technology Laboratories® was dissolved in 1000ml distilled water and mixed on hotplate then sterilization was done on 121 ºC in autoclave.
Appendix 3: Preparation of King’s B (KB) Medium
Ingredient Quantity Proteose Pepton 20 g
K2HPO4 1.5 g
MgSO4. 7H2O 1.5 g Agar 20 g Glycerol 15 ml Distilled Water 1000 ml
96
97
All the ingredients were mixed in 1000 ml distilled water with 15ml glycerol and was autoclaved at 15 psi and 121 ºC for 25 minutes.
Appendix 4: Preparation of Levan Media
Ingredient Quantity Nutrient Agar 28 g Sucrose 5% Distilled Water 1000 ml Nutrient agar was prepared by adding extra 5% sucrose and was autoclaved at 121 ºC for 25 minutes.
Appendix 5: Preparation of Oxidase Solution
Ingredient Quantity N,N,N',N'-Tetramethyl-p-phenylenediamine·2HCl 1 g Distilled Water 100 ml 1 g reagent was mixed in 100ml distilled water and was stored. Fresh solution was used for oxidase test.
Appendix 6: Preparation of Arginine dihydrolase Solution
Ingredient Quantity Peptone 1 g NaCl 5 g
K2HPO4 0.3 g Phenol Red 0.016 g Arginine 10 g Agar 3 g Distilled Water 1000 ml All the ingredients were suspended in 1000 ml distilled water and placed on hot plate to dissolve. Sterilization was done by autoclaving at 121 ºC.
98
Appendix 7: Preparation of Gelatine Media
Ingredient Quantity Yeast Extract 3 g Peptone 5 g Gelatin 120 g Distilled Water 1000 ml All the ingredients were dissolved in 1000 ml water and was autoclaved.
Appendix 8: Preparation of Aesculin Media
Ingredient Quantity Esculine 1 g Peptone 13 g Ferric citrate 0.5 g Yeast Extract 5 g Agar 15 g NaCl 5 g Distilled Water 1000 ml All ingredients were dissolved in 1000 ml distilled water and pH was maintained to 7.0
± 0.2 at room temperature.
Appendix 9: Preparation of Tyrosinase Media
Ingredient Quantity Casein hydrolysate 10 g
K2HPO4 0.5 g
MgSO4. 7H2O 0.5 g L-Tyrosin 1 g Agar 15 g Glycerol 15 ml Distilled Water 1000 ml
99
All ingredients were added in 1000 ml distilled water and was autoclaved.
Appendix 10: Preparation of L-Tartrate Media
Ingredient Quantity
NH4H2PO4 1 g
K2HPO4 1 g
MgSO4. 7H2O 0.2 g NaCl 5 g Agar 15 g Bromothymol blue 10 ml Sodium tartrate 2 g Distilled Water 1000 ml All the ingredients were dissolved in 1000 ml distilled water and was autoclaved at 121
ºC.