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ROLE OF NATTRASSIA SP. IN FRUIT ORCHARD DECLINE
AND DETERMINATION OF KEY FACTORS FOR ITS
MANAGEMENT
FAISAL SOHAIL FATEH 94-arid-224
Department of Plant Pathology Faculty of Crop and Food Sciences Pir Mehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018 2
ROLE OF NATTRASSIA SP. IN FRUIT ORCHARD DECLINE
AND DETERMINATION OF KEY FACTORS FOR ITS
MANAGEMENT
by
FAISAL SOHAIL FATEH (94-arid-224)
A thesis submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Plant Pathology
Department of Plant Pathology Faculty of Crop and Food Sciences PirMehr Ali Shah Arid Agriculture University Rawalpindi Pakistan 2018
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"In the Name of Allah, the most Beneficent, the most Merciful" 8
DEDICATION
To
MyAffectionate, Loving Parents
&
My Sweet Family
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CONTENTS
Page List of Tables xii List of Figures xvii List of Acronyms xx Acknowledgements xxi ABSTRACT xxii 1 GENERAL INTRODUCTION 1 1.1 EXPORT OF TARGET FRUITS 1
1.2 AREA AND PRODUCTION OF TARGET 2
ORCHARDS IN PAKISTAN
1.3 FRUIT FACING DECLINE 2 1.3.1 Citrus Decline 2
1.3.2 Mango Decline 4
1.3.2.1 Progressive or slow decline or dieback 4
1.3.2.2 Quick dieback 4
1.3.2.3 Mango sudden death (MSD) 5
1.3.2 Guava Decline 5 1.4 PATHOGENS INVOLVED IN DECLINE 6 1.4.1 Nattrassia mangiferae 6
1.4.2 Synonyms 6
1.4.3 Taxonomy 6
1.4.4 Host Range 7
1.4.5 Symptoms 7
1.4.6 Biology and Management 7
2 OCCURRENCE AND SYMPTOMATOLOGY OF CITRUS, 9 MANGO AND GUAVA DECLINE IN PUNJAB, PAKISTAN 10
2.1 INTRODUCTION 9 2.2 REVIEW OF LITERATURE 12 2.2.1 Decline of Fruit Orchards 12 2.2.1.1 Citrus decline 12 2.2.1.2 Mango decline 14 2.2.1.3 Guava decline 15 2.3 MATERIALS AND METHODS 17 2.3.1 Survey of Target Fruit Orchards for Disease Assessment 17 2.3.1.1 Profile of areas surveyed 18 2.3.1.1.1 Sargodha 18 2.3.1.1.2 Multan 18 2.3.1.1.3 Northern Irrigated Plan (Zone Iva) 19 2.3.2 Disease Incidence 35 2.3.3 Disease Severity 35 2.3.4 Disease Index 35 2.3.5 Symptomatology 36 2.4 RESULTS 36
2.4.1 Disease Assessment 36
2.4.1.1 Citrus decline assessment 36
2.4.1.2 Mango decline assessment 38
2.4.1.3 Guava decline assessment 47
2.4.2 Symptomatology 51
2.5 DISCUSSION 62
3 PREVALENCE OFNATTRASSIASP. FROM CITRUS, 64
MANGOAND GUAVA ORCHARDS ALONG WITH OTHER
DECLINE CAUSING FUNGI
3.1 INTRODUCTION 64
3.2 REVIEW OF LITERATURE 66 11
3.3 MATERIALS AND METHODS 68
3.3.1 Sampling Methodology 68
3.3.2 Isolation and Identification of Pathogens 69
3.3.2.1 Potato dextrose agar medium (PDA) 69
3.3.2.2 Malt extract agar (MEA) 69
3.3.2.3 Stem decoction glucose agar (SDGA) 69
3.3.2.4 Sterilization 70
3.3.2.5 Isolation from infected tissues 70
3.3.3 Microscopy 70
3.3.4 Determination of Fungal Frequency Percantage 71
3.3.5 Purification of Fungal Cultures 71
3.4 RESULTS 71
3.4.1 Plant Parts Wise Fungal Frequency Percentage 71
3.4.1.1 Mycoflora from citrus decline affected trees 71
3.4.1.2 Mycoflora from mango decline affected trees 72
3.4.1.3 Mycoflora from guava decline affected trees 72
3.5 DISCUSSION 93
4 ROLE OFNATTRASSIA MANGIFERAEAND 97 OTHERDECLINE CAUSING FUNGI IN SYMPTOMS DEVELOPMENT 4.1 INTRODUCTION 97 4.2 REVIEW OF LITERATURE 99 4.3 MATERIALS AND METHODS 101 4.3.1 Pathogenicity Tests 101 4.3.1.1 Flap method 101 4.3.1.2 Root injury method 101 12
4.4 RESULTS 103 4.5 DISCUSSION 111 5 STUDIES ON FACTORS AFFECTING DEVELOPMENT 114 OFFUNGI WITH SPECIAL REFERENCE TO NATTRASSIA SP. 5.1 INTRODUCTION 114 5.2 REVIEW OF LITERATURE 114 5.3 MATERIALS AND METHODS 117 5.3.1 Effect of Temperature, Light, pH and Culture Media 117 onFungal Growth 5.3.1.1 Effect of culture media 117 5.3.1.2 Effect of temperature 118 5.3.1.3 Effect of light 118 5.3.1.4 Effect of Ph 118 5.4 RESULTS 119 5.4.1 Effect of Culture Media on Radial Mycelial Growth 119 5.4.2 Effect of Temperature on Radial Mycelial Growth 120 5.4.3 Effect of Light on Radial Mycelial Growth 122 5.4.4 Effect of pH on Radial Mycelial Growth 122 5.5 DISCUSSION 129 6 MORPHOLOGICAL VARIABILITY AMONG ISOLATES 132 OFCOMMONLY FOUND FUNGUS (NATTRASSIA MANGIFERAE)FROM DECLINE AFFECTED CITRUS, MANGO AND GUAVA TREES 6.1 INTRODUCTION 132 6.2 REVIEW OF LITERATURE 134 6.3 MATERIALS AND METHODS 136 6.3.1 Fungal Isolates 136 6.3.2 Morphological Characterization 136 6.4 RESULTS 136 6.5 DISCUSSION 145 7 FACTORS FAVOURING THE DECLINE DISEASE 147 13
ANDDEVELOPMENT OF STRATEGIES FOR ITS MANAGEMENT 7.1 INTRODUCTION 147 7.2 REVIEW OF LITERATURE 148 7.3 MATERIALS AND METHODS 150 7.3.1 Surveys for Factor Favoring Decline in Fruit Orchards 150 7.3.2 Statistical Analysis 151 7.4 RESULTS 151 7.5 DISCUSSION 191 8 GENERAL DISCUSSION 198 8.1 ASSESSMENT OF DECLINE IN CITRUS, MANGO 198 AND GUAVA 8.2 COMMON SYMPTOMS OF DECLINE IN CITRUS, 201 MANGO AND GUAVA 8.3 MYCOFLORA ISOLATED FROM DECLINE 202 AFFECTED TREES PATHOGENICITY OF THE IMPORTANT DECLINE 203 8.4 CAUSING FUNGI 8.5 FACTORS AFFECTING THE GROWTH AND 203 DEVELOPMENT OF NATTRASSIA SP. 8.6 MORPHOLOGICAL CHARACTERISTICS OF 204 NATTRASSIA SP. FROM CITRUS, MANGO AND GUAVA SUMMARY 206 LITERATURE CITED 210
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List of Tables Table No. Page
2.1 Areas surveyed for citrus decline assessment in district Sargodha 22
2.2 Areas surveyed for mango decline assessment in various districts 25 of Punjab 2.3 Areas surveyed for guava decline assessment in various districts of 31 Punjab 2.4 Mean citrus decline incidence, severity and disease index 39 indifferent tehsils of district Sargodha 2.5 Mean citrus decline incidence, severity and disease index 39 indifferent location of tehsil Bhalwal of district Sargodha 2.6 Mean citrus decline incidence, severity and disease index 40 indifferent location of tehsil KotMomin of district Sargodha 2.7 Mean citrus decline incidence, severity and disease index 41 indifferent location of tehsil Sargodha of district Sargodha 2.8 Mean citrus decline incidence, severity and disease index 42 indifferent location of tehsil Sahiwal of district Sargodha 2.9 Mean citrus decline incidence, severity and disease index 43 indifferent location of tehsil Shahpur of district Sargodha 2.10 Mean citrus decline incidence, severity and disease index 43 indifferent location of tehsil Sillanwali of district Sargodha 2.11 Mean mango decline incidence, severity and disease index 46 indifferent districts of Punjab province 2.12 Mean mango decline incidence, severity and disease index 46 indifferent location of district Khanewal 2.13 Mean mango decline incidence, severity and disease index 48 indifferent location of district Multan 2.14 Mean mango decline incidence, severity and disease index 49 indifferent location of district Muzaffar Garh 2.15 Mean mango decline incidence, severity and disease index 49 indifferent location of district Rahim Yar Khan 15
2.16 Mean mango decline incidence, severity and disease index 50 indifferent location of district Bahawalpur 2.17 Mean guava decline incidence, severity and disease index 53 indifferent districts of Punjab province 2.18 Mean guava decline incidence, severity and disease index 53 indifferent location of district Kasur 2.19 Mean guava decline incidence, severity and disease index 54 indifferent location of district Sheikhupura 2.20 Mean guava decline, severity and disease index in 55 differentlocation of district Nankana Sahib 2.21 Decline symptoms development percentages in citrus, mangoand 61 guava trees 4.1 Response of citrus plants against artificial inoculation of N. 105 mangiferaeandB. theobromaeusing flap method 4.2 Response of citrus plants against artificial inoculation of N. 105 mangiferaeandB. theobromaeusing root injury method 4.3 Response of mango plants against artificial inoculation 106 ofCeratotocystissp.,N. mangiferae,B. theobromaeandFusariumsp. using flap method 4.4 Response of mango plants against artificial inoculation 107 ofCeratotocystissp.,N. mangiferae,B. theobromaeandFusarium sp. using root injury method 4.5 Pathogenicity of fungi associated with guava decline by usingflap 108 method inoculation 4.6 Pathogenicity of fungi associated with guava decline by usingroot 109 injury method of inoculation 5.1 Effect of culture media on radial mycelial growth of the 121 fungiisolated from Citrus on PDA medium 5.2 Effect of culture media on radial mycelial growth of the 121 fungiisolated from mango on PDA medium 5.3 Effect of temperature on radial mycelial growth of the fungi 123 isolated from guava on PDA medium 16
5.4 Effect of temperature on radial mycelial growth of the fungi 123 isolated from citrus on PDA medium 5.5 Effect of temperature on radial mycelial growth of the 124 fungiisolated from mango on PDA medium 5.6 Effect of temperature on radial mycelial growth of the 124 fungiisolated from guava on PDA medium 5.7 Effect of light on radial mycelial growth of the fungi isolated from 125 citrus on PDA medium 5.8 Effect of light on radial mycelial growth of the fungi isolatedfrom 125 mango on PDA medium 5.9 Effect of light on radial mycelial growth of the fungi isolatedfrom 127 guava on PDA medium 5.10 Effect of pH on radial mycelial growth of the fungi isolated 127 fromcitrus on PDA medium 5.11 Effect of pH on radial mycelial growth of the fungi isolated 128 frommango on PDA medium 5.12 Effect of pH on radial mycelial growth of the fungi isolated 128 fromguava on PDA medium 6.1 Morphological characteristics of N. mangiferae isolates fromcitrus 138 in tehsil and district Sargodha 6.2 Morphological characteristics of N. mangiferae isolates fromcitrus 139 in tehsil Bhalwal, district Sargodha 6.3 Morphological characteristics of N. mangiferae isolates 140 frommango in district Multan 6.4 Morphological characteristics of N. mangiferae isolates 141 frommango in district Rahimyar Khan 6.5 Morphological characteristics of N. mangiferae isolates fromguava 142 in tehsil Pattoki of district Kasur 6.6 Morphological characteristics of N. mangiferae isolates fromguava 143 in tehsil Sharaqpur of district Sheikhupura 7.1 Factor affecting disease index (%) in selected orchards of citrusin 152 district Sargodha 17
7.2 Correlations of factors causing citrus decline disease index (%) 162 7.3 Correlation between citrus tree age groups with disease index(%) 164 7.4 Correlation between citrus tree age groups with disease index(%) 164 7.5 Correlation between mode of irrigation in citrus orchards 165 withdisease index (%) 7.6 Correlation between nutrition in citrus orchards with diseaseindex 165 (%) 7.7 Correlation between pruning in citrus orchards with diseaseindex 166 (%) 7.8 Correlation between ploughing under tree canopy in citrusorchards 166 with disease index (%) 7.9 Correlation between plant protection in citrus orchards withdisease 167 index (%) 7.10 Factors affecting decline disease index in selected orchards 168 ofmango growing districts in Punjab 7.11 Correlations of factors causing mango decline disease index (%) 174 7.12 Correlation between age group in mango orchards with 176 diseaseindex (%) 7.13 Correlation between intercropping in mango orchards withdisease 176 index (%) 7.14 Correlation between irrigation mode in mango orchards 177 withdisease index (%) 7.15 Correlation between ploughing under tree canopy in citrusorchards 177 with disease index 7.16 Correlation between pruning of trees in mango orchards 178 withdisease index (%) 7.17 Correlation between ploughing under tree canopy in 178 mangoorchards with disease index (%) 7.18 Correlation between plant protection in mango orchards 179 withdisease index (%) 7.19 Factors affecting decline disease index in selected orchards 181 ofguava growing districts in Punjab 18
7.20 Correlations of factors causing guava decline disease index (%) 185 7.21 Correlations of factors causing guava decline disease index (%) 187 7.22 Correlation between intercropping in guava orchards withdisease 187 index (%) 7.23 Correlation between irrigation mode in guava orchards withdisease 188 index (%) 7.24 Correlation between nutrition application in guava orchards 188 withdisease index (%) 7.25 Correlation between pruning of trees in guava orchards 189 withdisease index (%) 7.26 Correlation between ploughing under tree canopy in 189 guavaorchards with disease index (%) 7.27 Correlation between plant protection in guava orchards 190 withdisease index (%)
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List of Figures Figure No. Page 2.1 Agro-ecological zones 21
2.2 Areas surveyed for citrus decline in district Sargodha, Punjab, 24 Pakistan 2.3 Areas surveyed for mango decline in district Khanewal, Punjab, 27 Pakistan 2.4 Areas surveyed for mango decline in district Multan, Punjab, 28 Pakistan 2.5 Areas surveyed for mango decline in district Muzaffar Garh, Punjab, 29 Pakistan 2.6 Areas surveyed for mango decline in district R. Y. Khan, Punjab, 30 Pakistan 2.7 Areas surveyed for guava decline in district Kasur, Punjab, Pakistan 32 2.8 Areas surveyed for guava decline in district Sheikhupura, Punjab, 33 Pakistan 2.9 Areas surveyed for guava decline in district Nankana Sahib, Punjab, 34 Pakistan 2.10 Twig dieback in citrus 56 2.11 Bark Splitting and gummosis in citrus 56 2.12 Canker development in citrus 57 2.13 Citrus tree mortality 57 2.14 Bark splitting and oozing of thick darkish brown liquid in Mango 58 2.15 Gummosis and stem canker in mango decline affected tree 58 2.16 Mango Sudden Death affected dead tree (MSD) 59 2.17 Bark splitting and gummosis in decline affected guava tree 59 2.18 Tree canker in decline affected guava tree 60 2.19 Decline affected dead guava tree 60 3.1 Plant parts wise fungal frequency percentage from declineaffected 74 citrus trees at Chak No. 26/NB tehsil Bhalwal, Sargodha 3.2 Plant parts wise fungal frequency percentage from declineaffected 75 20
citrus trees at Chak No. 8 NB, tehsil Bhalwal of districtSargodha 3.3 Plant parts wise fungal frequency percentage from declineaffected 76 citrus trees at Chak No. 10 N/B, tehsil Bhalwal of districtSargodha 3.4 Plant Parts wise fungal frequency Percentage from declineaffected 77 citrus trees at Chak No. 56/NB of tehsil and districtSargodha 3.5 Plant parts wise fungal frequency percentage from declineaffected 78 citrus trees at Chak No. 112/NB of tehsil and districtSargodha 3.6 Plant parts wise fungal frequency percentage from declineaffected 79 citrus trees at Chak No. 27 SB of tehsil and district,Sargodha 3.7 Plant parts wise fungal frequency percentage from declineaffected 80 mango trees in BastiNandla, Multan 3.8 Plant Parts wise fungal frequency Percentage from declineaffected 81 mango trees in ChahNizamWala, Multan 3.9 Plant parts wise fungal frequency percentage from declineaffected 82 mango trees in Basti Band Bosan, Multan 3.10 Plant parts wise fungal frequency percentage from declineaffected 83 mango trees at Chak 22A, tehsil Liaqatpur of districtRahim Yar Khan 3.11 Plant parts wise fungal frequency percentage from declineaffected 84 mango trees at Chak 2P, tehsil Khanpur of district RahimYar Khan 3.12 Plant parts wise fungal frequency percentage from declineaffected 85 mango trees at MianwaliQureshian, tehsil and districtRahimYar Khan 3.13 Plant parts wise fungal frequency Percentage from declineaffected 86 guava trees at Sehjowal, tehsil Pattoki of district Kasur 3.14 Plant parts wise fungal frequency percentage from declineaffected 87 guava trees at Faizpur, tehsil Ferozewala of districtSharaqpur 3.15 Plant parts wise fungal frequency percentage from declineaffected 88 guava trees at AddaPulTorian of tehsil and districtNankana Sahib 3.16 Culture and macrospores of Fusarium sp. 89 3.17 Culture and conidia of Ceratocystis sp. 90 3.18 Cultural and conidia of Lasiodiplodia theobromae 91 21
3.19 Culture and spores of Nattrassia mangiferae 92 4.1 Flap method of inoculation. 110 4.2 Root injury method of inoculation. 110 6.1 N. mangiferaearthrospores from citrus. 144 6.2 N. mangiferae arthrospores from mango. 144
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LIST OF ACRONYMS
A Absent
Av. Average
BE Bhalwal East
BW Bhalwal West
CJA Carrot Juice Agar
D.I. Khan DeraIsmial Khan
Eds. Editors
GOP Government of Pakistan
J. Journal
MEA Malt Extrat Agar
MSDS Mango Sudden Death Syndrome
N Total number of plants
NARC National Agricultural Research Centre
P Present
Pak. Pakistan
PARC Pakistan Agricultural Research Council
PDA Potato Dextrose Agar
PI Pattoki
R.Y. Kan Rahimyar Khan
Sar Sargodha
SDGA Stem Decoction Glucose Agar
SP Sharqpur SW Silanwali ACKNOWLEDGEMENTS
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I would like to acknowledge my supervisor Prof. Dr. Tariq Mukhtar for his constant support, patience, sympathetic attitude, inspiring guidance, constructive criticism, timely advice and enlightened supervision during my research work and in the accomplishment of this manuscript.
Thanks to my respected teachers Prof. Dr. Muhammad Irfan Ul Haque and Prof.
Dr. Abdul Rauf Chauhdary for their helpful advice,inputs and encouragement during the course of my research studies and presentation of this manuscript.
I am deeply thankful to Dr. Munawar Raza Kazmi for his support and brilliant ideas to meet the objectives of my research.
I would specially like to extend my deepest gratitude to my parents, my beloved wife and my children for their prayers always remain with me.
Appreciation for my friends Dr. Muhammad Zakria, Dr. Atif Jamal, Mr.
Usman Raja, Dr. ShahzadAsad, Dr. Hussain Shah Hayatullah Tareen and my officers Dr. Iftikhar Ahmad, Dr. Anjum Munir and Dr. Shahid Hameed who always encouraged me for thecompletion of my Ph. D.
Finally, I would like to express my deepest gratitude to Ms.
ShumailaIqbal, Dr. TahiraYasmin and Dr. IshaqueMastoi who always backed me up to complete my research in time.
(FAISAL SOHAIL FATEH)
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ABSTRACT
Decline of citrus mango and guava is a serious threat in Pakistan. This study was undertaken to know the role of Nattrassia sp. in fruit orchard decline, its biology and listing down the management strategies. Its role was studies through recording the disease incidence and severity from citrus, mango and guava orchards in Punjab from Sargodha, Bhalwal, Multan, Rahimyar Khan, Pattoki,
Sharqpur and Ahmedpur. Disease prevalence was found in all of the above areas of citrus mango and guava with less or more intensities. The sampling from target tree crops resulted in almost similar type of mycoflora except Ceratocystis sp. was isolated from only decline affected mango trees. Other fungi include Nattrassia mangiferae, Lasiodiplodia theobromae, Botryosphaeria sp., Fusarium sp.,
Cladosporium sp., Aspergillus sp., Penecillium sp. andCurvularia sp. with different intensities from affected twigs, branches, stem at collar region and roots. However, the fungus Nattrassia sp. and Lasiodiplodia sp. were the most common fungi.
Pathogenicity was conducted to know the four decline causing fungi including
Nattrassia sp. It was found that Nattrassia sp. alone cannot cause tree mortality but contributes towards the mortality in combination with other fungi. The fungus
Nattrassia isolated from citrus is morphologically and physiologically different from isolates of mango and guava. However, there is resemblance in isolates of mango and guava. Most of the factors that favored the disease include no concept of pruning; faulty intercropping; flooding of the orchards; application of partially decomposed farm yard manure; poor nutrition; inadequate plant protection measures and less interest of the growers. The orchards can be managed by 25
adopting pruning, following recommended plant protection plans, nutrition based on leaf, soil and water sampling; avoiding plowing under tree canopies; if necessary to intercrop choose friendly crops. In short the decline of citrus, mango and guava have almost common, pathogens, common symptoms, common biology and hence the common management strategies may be adopted to avoid them in future.
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Chapter 1
GENERAL INTRODUCTION
Fruit orchards e.g. apple, pear, peach, grapes, citrus, mango and guava around the world are facing decline. Symptoms of decline vary with the kind of fruit orchards as well as the nature of causal organisms involved. The causal agents of decline reported from around the world include fungi, bacteria, nematode, phytoplasma, viruses and some nutrient deficiencies (Agrios, 2005).
In Pakistan important fruit trees such as citrus, mango, guava etc are facing decline. Among these fruits, citrus and mango decline have been addressed strongly as they are important foreign exchange earnings and export items. Mango is being given attention in all aspects but citrus needs special attention. Due to poor quality citrus in Pakistan we have been restricted by export markets. According to
Pakistan Horticulture Export Board, 20% of the citrus fruit is gone waste either due to diseases or post harvest mishandling (Humayun et al., 2005; Khanzada et al.,
2004). Other fruit trees like guava and loquat are also very famous fruit of Pakistan and are liked by all generations (Misra and Pandey, 2000).
1.1. EXPORT OF TARGET FRUITS
Pakistan is famous for citrus production especially for its kinnow and oranges. It ranks sixth in the world with 2.3 million tons production. Pakistan produces 95% of the world's Kinnow. According to an estimate, the country exported kinnows amounting to Rs. 15 million thousands in year 2012. By the year
2013 the citrus export target per anum was set a 500,000 tones within the next fiveyears, and expecting an export income of $300 million but due to some
1 27
keyconstraints that needed to be addressed these ambitious targets were not achieved (Anonymous, 2013).
Mango ranks second after citrus in Pakistan. It is making its way to the foreign market by its taste and shapes. The main foreign markets include
United Kingdom, France and Germany which consumes 15 % of mangoes imported by the EU. Imports of Pakistani mango has increased since 2002 by EU countries up to 50% i.e. 199,000 tons worth 228 million euros in 2006. The reason for this growth is that mangoes are not grown in Europe. The major export of mango is done by India, Mexico, Brazil and Peru. Pakistani mango has a substantial export potential in foreign markets which has not been realized so far.
Mango exports from Pakistan reached up to Rs. 3.27 million in 2012 (Anonymous,
2013).
1.2.AREA AND PRODUCTION OF TARGET FRUIT ORCHARDS INPAKISTAN
Citrus, mango and guava are grown on an area of 0.193; 0.175 and
0.064million hectares respectively with the production of 2.40; 1.720 and 0.490 million tons respectively (Anonymous, 2016).Citrus orchards vary from less than
10 heactares to 400 hectares (Ashraf et al., 2015), mango orchards range in size from less than 2 hectares to 200 hectares (Khan, 2010) while guava orchards range from less than hectare to 96 hectare (Khushk et al., 2009).
1.3. FRUITS FACING DECLINE
1.3.1. Citrus Decline 28
Citrus decline sometimes also referred to as “Citrus Dieback” which is not a specific disease but is symptomatic expression of many disorders in the plant. The malady is also termed as „frenching‟ or „chlorosis (Anonymous, 2000).
Citrus species which are being cultivated in Pakistan include grape- fruit (Citrus paradisi Macfad.), mandarin (Citrus reticulata Blanco), sweet
Orange (Citrus sinensis (L.) Osbeck), bitter Orange (Citrus aurantium L.),
Lime (Citrus aurantifolia (Christm.) Swingle), Rough Lemon (Citrus jambhiri Lush.) and Kinnow (it is a Hybrid of Citrus nobilis and Citrus deliciosa).
The kinnow is grown largely in citrus orchards of Pakistan but not a single variety has shown resistance against decline (Anonymous, 2013).
Scientists during 1970s and 1980s reported that relatively large number of trees were declining and showed dieback in all the citrus areas visited both on mandarin and on sweet orange trees. Thus it appears that the decline affecting citrus is not new. Young decline affected citrus trees grow well initially, and then begin to show symptoms only after three or four years. (Chapot, 1975; Boveet al.,
1983).
Symptoms of decline included overall pale green-yellowish colour of foliage; mature leaf drop and replacement of normal-sized leaves by small ones; an upright position of small leaves along shoots; zinc-deficiency symptoms on small leaves but without green islands on an overall yellow blade; mild leaf mottle on old leaves; dieback and stunting (Chapot, 1975). 29
Citrus decline is known to be caused by biotic (fungi, nematodes, viruses, phytoplasma etc.) and abiotic factors (soil, nutritional and rootstock factors).
Similarlysingle variety cultivation has further aggravated disease situation in existing citrus gowing areas. It has been observed that decline is much more prevalent in high rainfall receiving areas. Besides the local diseases, diseases of international importance like citrus tristeza virus (CTV) and citrus greening have been reported from the existing citrus growing areas and contribute to citrus decline. (Naqvi, 2004).
1.3.2. Mango Decline
In case of mango, decline has different forms, generally known as progressive, slow decline or die back; quick die back and sudden death (Fateh et al., 2006). The details are given as below.
1.3.2.1. Progressive or slow decline or dieback
In progressive decline, twig and branch dieback is seen on mango trees. It starts from top of the canopy and progresses towards the lower braches.This affect the normal flush of trees which are supressed as well as cause leaf disoloration.
Due to weak flushes the canopy seems barren and dead twigs are generally high in number. This kind of situation is mostly prevalent in poorly managed orchards where proper nutrition, plant protection and irrigation practices are neglected. Such kind of decline can be easily managed if cultivating orchards according to best available production technology (Fateh et al., 2009).
1.3.2.2. Quick dieback 30
In this kind of dieback, trees start losing their shape quickly. Suck kind of dieback is the indication of either deficiency or toxicity of some elements. Leaves change colour from green to yellow and drop; the moisture does not reach the higher branches and trees' tops look dead. This kind of drought situation allows the opportunistic fungi to invade the plants and in response the plants produce gum of various colors. The appropriate nutrition, irrigation at required timing as well as foliar sprays of sustemic fungicides help in managing the quick dieback of mango trees (Kazmi et al., 2007).
1.3.2.3. Mango sudden death (MSD)
This kind of decline is alarming and is a real threat for the mango growers.
The name "sudden death" given to this disease is tree death visible inshortest time may be in few days or a month. Once the decline causing pathogens infect trees the internal break down of the tissues is initiated. However, the symptoms remain masked. As the environment becomes favourable for the rapid growth of pathogen and trees lose vigor, healthy looking trees show severe symptoms resulting in sudden mortality. The symptoms include leaf drying, drooping and sometimes become leathery. The cracks appear on the stem at collar region, bark splits and trees ooze dark brown gum. Sometimes thick dark brown liquid is released from the colar region. In the infected trees when bark is removed from the stem, mouse grey streaks appear which show that pathogens have fully invaded the trees and its recovery is unlikely (Fateh et al., 2006).
1.3.3. Guava Decline
Guava Decline has been observed in the areas of sub continent in 1935 before partition. However, it was reported in 1947 for the first time (Naqvi, 2004). 31
The symptoms of guava decline include leaf discoloration, twigs necrosis, branch splitting, drying of branches mostly at one side of the trees and irregular tree symmetry. On removal of bark, the stem shows canker. Several fungi have been reported from guava decline like Lasiodiplodia theobromae, Fusarium sp.,
Botryosphaeria sp. Phytophthora sp. etc. (Bukhari, 2009).
1.4.PATHOGENS INVOLVED IN DECLINE
Decline in different forest and fruit trees involve different pathogens e.g. fungi such as Ceratocystis fimbriata, Armillaria sp., Nattrassia magniferae,
Lasiodiplodia theobromae, Phoma sp., Phomopsis sp., Fusarium sp., Viruses such as citrus tristeza virus (CTV), Nematodes especially in case of citrus decline
(Tylenchulus semipenetrans) and in mango (Hemicriconemoides, Longidorus sp.), bacteria such as Pseudomonas sp. and Phytoplasma. Among these pathogens, the fungus Nattrassia magniferae has been reported from almost all of the forest and fruit trees facing decline (Fateh et al., 2006; Bukhari, 2009).
1.4.1. Nattrassia magniferae
1.4.1.1. Synonyms
This fungus has different synonyms e.g. Dothiorella mangiferae,
Exosporina fawcettii, Fusicoccum dimidiatum (Penz.), F. eucalypti, Hendersonula agathidis, H. cypria Nattrass, H. toruloidea Nattrass, Neofusicoccum mangiferae,
Neoscytalidium dimidiatum, Scytalidium dimidiatum, S. lignicola and Torula dimidiate (Farr et al., 2005).
1.4.1.2. Taxonomy 32
The Nattrassia mangiferae belongs to Phylum Ascomycota; Class
Ascomycetes; Sub ClassDothidiomycetes; FamilyBotryosphaeriacea; Genus
Nattrassia and Species: N. mangiferae(Farr et al., 2005).
1.4.1.3. Host range
In fruit trees mango, guava, citrus, apple, plum, mulberry, fig and walnut are the hosts. In forest trees the recorded hosts are Arbutus sp., Euclyptus sp.,
Madrone sp.Besides causing different plant diseases, this fungus is equally harmful to human beings causing skin diseases (Atta and Aref, 2013).
1.4.1.4. Symptoms
The fungus Nattrassia magniferae causes different symptoms in different trees e.g. dieback, cankers, blossom blight, wilt, necrosis, decline and death. As mentioned above the symptoms of slow or quick die back can result from infection by the fungus N. mangiferae. The symptoms are confused with those produced by other pathogens such as Botryodiplodia theobromae and Botryosphaeria sp.Brownish streaks appear when the bark is removed from branches and stem as well.The trees are apparently healthy, with masked symptoms and produce rotten fruits (Farr et al., 2005).
1.4.1.5. Biology and management
A very few scientists made efforts to study the biology of this potential pathogen which is involved in many of the decline diseases in fruits and forest trees. To devise effective strategies for its management,biology of N. mangiferaewas studied in South Africa. In this regard many chemicals were applied in preharvest stage of the trees to see their effect on management of pre and 33
post harvest diseases of citrus fruit. Among all thetested fungicides, pre-harvest applications of copper-oxychloride on monthly basis during fruit set till harvest significantly managed the disease (Saimaan, 1997).
Keeping in view importance of the fungus associated with decline of fruit trees, lack of information on its biology and management, the present studies were designed with the following objectives:
Determination of prevalence of Nattrassia sp. in target orchards (mango, citrus
and guava) of Punjab.
Studies on the biology and epidemiology of Nattrassia sp. causing decline in
fruit orchards and
Determination of factors causing tree decline for the management of Nattrassia
sp.
34
Chapter 2
OCCURRENCE AND SYMPTOMATOLOGY OF CITRUS, MANGO AND GUAVA DECLINE IN PUNJAB, PAKISTAN
2.1. INTRODUCTION
Pakistan is an agricultural country. The Gross Domestic Product (GDP) of
Pakistan was 20.9 % in 2014-15. Livelihood of 43.5 % of rural population exists in agriculture.Enhance productivity and less vulnerability are the main objectives of food security. To increase economic growth and reducing poverty in Pakistan through agriculture, a policy framework coupled with best socio political climate, good governance is fundamental. The government of Pakistan is focusing on horticulture, livestock and fisheries. Along with developed nations efforts have been made to improve production technologies and linking farmers directly with markets and industry (Anonymous, 2015).
Horticulture is the main hub of agricultural economy. Approximately12 million tons of fruits, vegetables and spices are produced on yearly basis. Among the important fruits citrus contributes2,000,000, mangoes 1,000,000, dates63000 and apples 400,000 tons annually. There are few minor fruits which are harvested round the year in different geographical zones of Pakistan.
Citrus, mango and guava are grown on an area of 190,000,170,000 and
63000 hectares respectively with the productionsof 240,000, 1,720,000and 490,000 tons respectively (Anonymous, 2016).
Citrus is leading the fruits industry in Pakistan and occupies first position.
Pakistan produces approximately 3 to 4 % of the world citrus and the exports are
9 35
limited to only about 0.8 % of harvest. It is a known fact thatup to 95 % of the world's kinnow is produced in Pakistan. In Punjab 85% area under citrus is in tehsil
Sargodha and Bhalwal. The rest of 15% is shared by Sillanwali, Sahiwal and
Sahpur tehsils. However, Bhalwal is leading in terms of citrus cultivation. Kinnow is the famous variety in these areas (Sharif and Waqar, 2005).
In Pakistan, citrus has been planted in the new areas but the average economic life of citrus is still not comparable with other citrus producing countries.
Fifty years is the average economic life of citrus all around the world. However, the average life of citrus in Pakistan is about 25 years and trees start to decline after
7-10 years of production (Chaudhry, 2003).
Citrus decline in Pakistan has been thought to be found since 1960s from the Khyber Pakhtoonkhwah province. However, it was reported in 1970 in the form of dieback and the causes were attributed to deficiency symptoms and drainage problems in the orchard (Chapot, 1970).
In citrus, particular decline symptoms are wilt and chlorosis of leaves followed by leaf and fruit drop. Affected branches show die back starting from top to the lower branches, a healthy tree suddenly wilts, turns yellow and dies rapidly
(Safdar et al., 2010).
Mango decline in Pakistan was reported from the Punjab province in one of the orchards at Muzaffar Garh in 1995. The symptoms like leaf drooping, bark splitting, gummosis, stem canker and stem bleeding appeared. This type of decline was called quick decline. Twig dieback and the branches dieback are also observed but that might be due to many reasons (Fateh et al., 2006; Naqvi, 2004). 36
The association of pathogenic fungi and role of bark beetle in mango decline has been studied in Pakistan as well as few other countries where the disease has become a dilemma. The most common symptoms produced were leaf drying, discoloration gum oozing, cankers development under the bark and vascular blockage. The symptoms intensify when Hypocryphalus mangiferae,commonly known as bark beetle bores in the stem. Some of local mango varieties of Pakistan like Malda, Langra and Anwar Ratol show a little bit tolerance to decline.The mycoflora from decline affected treesand the bark beetle
(H. mangiferae) includedLasiodiplodia theobromae, Ceratocystis fimbriata and
Phomopsis sp. (Masood et al., 2011).
Guava decline have been found existing in India since 1935 but it wasreported for the first time in 1947. Severely decline affected trees show leaf drooping and discoloration, dead twigs and rotted, discolored stems. The branches die on one side of the trees initially and later cover the entire canopy. The tree mortality occurs with complete defoliation and dry wood.
During a survey of guava growing areas in Sheikhupura district of Punjab,
Pakistan maximum disease incidence was recorded in Tehsil Sharaqpur followed by Pattoki of district Kasur (Safdar et al., 2015).
As orchards in the Punjab province are declining, trees are becoming debilitated and less productive, therefore, the first step was planned to conduct surveys in major areas to determine the incidence and prevalence so that proper management measures might be suggested to the farmers.
The objectives of this study were as under: 37
1) To assess the disease occurence and severity in the orchards of
citrus, guava and mango from some selected locations famous for the
production of the commodity in the province of Punjab.
2) To find out the similarity of symptoms of citrus, mango and guava
decline affected trees.
2.2. REVIEW OF LITERATURE 2.2.1. Decline of Fruit Orchards
Many fruit orchards in Pakistan have prevalence of decline disease including citrus, mango, guava, loquat and pomegranate. However, special importance is given to citrus, mango and guava decline being the most important fruit crops of Pakistan.
2.2.1.1. Citrus decline
Citrus decline is a global problem in citrus growing areas. Mostly the mature trees become victim of decline. The recovery of an infected tree is quite difficult. Twig blight of citrus is mostly confined to tree canopies but its symptoms resemble with other internationally important diseases like greening, CTV and slow decline (caused by nematode Radophilus citrophilus) in Florida (EPPO/CABI,
1996a, b and c). The decline affected citrus trees show zinc deficiency. The significance of high levels of zinc is not known but its analysis may be helpful for diagnosis of decline (Young et al., 1980a and b).
Other than apparent symptoms, there is also change in tree physiology.
Studies have shown that in decline affected citrus trees water conductivity in the xylem is impaired. The trees failed to intake water and food because the xylem was 38
blocked by light-yellow dark-brown filamentous plugs known as tylosis. The dieback symptoms appear due to lack of water transport to the canopy (Brlansky et al., 1985).
Most of the scientists thought that it was citrus tristeza virus which caused citrus decline and they did research with this vision. A survey was conducted in citrus growing areas of Khyber Pakhtunkhwah province of Pakistan to assess various citrus diseases. Most common diseases in citrus orchards and nurseries included citrus tristeza, citrus variegation, citrus exocortis, citrus cachexia
(xyloprosis) and citrus greening. High incidence of these devastating pathogens has caused the severe citrus decline, drastic yield and quality losses in citrus fruits in the region (Arif et al., 2005).
Another school of thought said that phytoplasma and bacteria are involved in citrus decline. Burney et al. (2007) during a survey of citrus growing areas in
Punjab found that citrus decline was a result of combination of bacteria and phytoplasma. The leaf sample showed that Xanthomonas compestris is the most prevalent bacterium with 50% incidence in Jhang, 17.24% in Sahiwal and no incidence was found in Faisalabad, Toba Tek singh and Kasur. Phytoplasmal disease incidence was found to be 27.58% in Sahiwal, 15.62% in Toba Tek Singh while it was not encountered in Jhang.
The fungal involvement in citrus decline was found in Oman as well.
During survey conducted in various citrus growing districts, it was observed that citrus varieties i.e. acid limes and sweet limes exhibited gummosis at collar region.
The isolation from bark and roots resulted in 19 fungal species. Among these the 39
most common were Lasiodiplodia hormozganensis, L. theobromae and Fusarium solani. On artificial inoculation of these common fungi, acid lime and sweet lime seedlings reproduced decline and gummosis symptoms. The nursery studies on sweet lime showed association of 12 fungal species, which is evidence that nurseries act as a main source for some citrus pathogens (Al Sadi et al., 2010).
During a survey in Pakistan from citrus growing districts especially
Sargodha and Faisalabad, decline incidence was found higher in Sargodha except
Sillanwali and Kot Momin tehsils. The isolated fungi from decline affected tree tissue and root samples were Fusarium semitectum, F.solani, Helminthosporium sp. and Phytophthora sp., with varying frequencies. (Safdar etal., 2010).
Nematologists said that these are nematodes causing citrus decline.
Khanzada et al. (2007) conducted surveys in different districts of Punjab and collected roots and soil samples from the orchards showing decline symptoms. In all areas the incidence of Tylenchulus semipenetrans, the cause of slow decline was the maximum (56%) in variety Feutrell‟s Early followed by 40% in lemon and orange, 35% in Kinnow and Musambi. The minimum incidence (20%) was observed in grape fruit. A survey in Punjab showed that the nematode (T. semipenetrans) population was recorded above economic threshold level in all citrus growing tehsils of Sargodha districts and this might also contributing towards citrus decline (Safdar etal., 2010).
2.2.1.2. Mango decline
Mango decline is prevalent in the world especially in Brazil, Oman,
Pakistan and India. A fungus Diplodia theobromae associated with sudden decline 40
of mango for the first time was reported in Oman. During 1999 up to 60% of trees in parts of the Al Batinah region had died of strange wilt disease. Trees showed gummosis, wilting, browning of leaves and died in 5-6 months after appearance of first symptoms. Dead wood was dark brown. It was thought that a bark beetle
(Cryphalus scabrecollis), is responsible for spread of the disease (Al-Adawi et al.,
2002).
The etiology of mango sudden death in Pakistan was first time reported in
Pakistan during 2005. Incidence of the disease was high in mango growing areas of province Sindh as compared to the Punjab province. The mycoflora isolated included Botryodiplodia theobromae, Phytophthora sp., and Fusarium sp. In the mean time a new fungus was reported in Oman during 2006, known as
Ceratocystis fimbriata which attacked mango in combination with L. theobromae.
Later it was reported new species Ceratocystis omanensis (Al- Adawi et al., 2006).
In Pakistan the fungus C. fimbriata for the first time was reported in Sindh from declining mango trees. It's identification was confirmed by morphological characteristics of perithecia (brown to black with globose base, necks almost 800-
900 µm long with ostiolar hyphae), ascospores (elliptical 4-8 × 2-5 µm, hat shaped) conidiophores (hyaline, septate up to 150 µm long and conidia that were cylindrical, sometimes in chains and truncated at the ends (Fateh et al., 2006).
2.2.1.3. Guava decline
Guava decline is also termed as guava wilt; Rosemary (2001) reported that in guava decline leaves start wilting from the top of the tree. Sometimes, it is a rapid decline and sometimes it is a slow decline. There is general wilting of all the 41
leaves. The leaves become yellow or purple and drop. During the initial stages, the leaves dry out and often remain on the tree. If there are fruit on the tree, fruit development ceases immediately, and you get mummified fruit on the tree. Guava wilt disease killed off orchard trees randomly, and at the end, there would be no orchard left. The original research concentrated on trying to control the disease with chemicals, but was totally ineffective. There are no chemicals available to control the disease.
Guava decline is distributed in many countries of the world. Most of the guava is cultivated in India (where it is known as poor men‟s apple) and Pakistan.
Guava decline in India is considered a soil borne disease associated with association of several pathogens. Among most common pathogens, Fusarium oxysporum f. sp. psidii and F. solani were reported. A new potential fungus
Gliocladium roseum was also identified. Heavy losses in guava production in India have been recorded due to this disease (Misra, 2007).
In South Africa guava decline has been reported to cause by a fungus identified as Penicillium vermoesenii. Earlier, the fungus has been associated with blight of ornamental palms in the United States and Belgium. It was later reported from guava in the above mentioned countries. The Koch's postulates confirmed the pathogenicity of the fungus in the glasshouse and field conditions by producing symptoms like wilting, chlorosis and defoliation in nursery seedling as well as branches of mature trees (Schoeman et al., 2008).
Avelar-Mejia et al. (2003) described the effect of guava tree decline on the anatomy of branches and roots of guava. Guava tree decline in Calvillo, 42
Aguascalientes and Apozol, Zacatecas, Mexico did not affect the anatomy of the branches of the tree. The disease was associated with root histological alterations caused by Meloidogyne sp. infestation. The bark, phloem and vascular cambium tissues showed hyperplasia and hypertrophy, as well as an increase in polyphenol content and necrosis. The decline was related with an increase in lignin and polyphenol content, and with the reduction in root starch. Fungi were observed in the xylem and cambium of severely affected trees, although the genus and species were not determined. The partial plugging of xylem vessels was not related with the decline.
During a survey in one of the guava growing districts of the Punjab province of Pakistan i.e. Sheikhupura, decline disease incidence was reported as high as 36% inTehsil Sharaqpur. The isolated fungi included B. theobromae,
Fusarium oxysporum f.sp. psidii, Phytophthora parasitica, F. solani,
Helminthosporium sp. and Curvularia lunata. The pathogenicity of most dominating fungus (B. theobromae) was confirmed on green house potted guava plants of 2 years (Safdar et al., 2015).
2.3. MATERIALS AND METHODS
2.3.1.Survey of Fruit Orchards for Disease Assessment
According to world map Pakistan is located between 24o to 37o latitudes in north and 61o to 75o longitudes in east. It is spread over 1600 kilometres from north to south and from east to west 885 kilometres. The total area of the country is 796,
095 square kilometres. Pakistan has best climate for agriculture ranging from subtropical to semi-arid. The per anum rainfall ranges from 125 mm (extreme southern plains) to 900 mm in the mountainous and northern plains. Monsoon 43
season is known for heavy rainfall and receives 70 % of the total rainfall in generarlly from month of July to September. The remaining 30 % rainfall is in winter. In plains summers is extreme with maximum temperature more than 40 °C, while the temperature in winter is a few degrees above the freezing point.
2.3.1.1. Profile of areas surveyed
A brief description of the areas surveyed for incidence and prevalence of decline is given below.
2.3.1.1.1. Sargodha
Sargodha is known as Pakistan's best citrus-producing area. It is situated
220 kilometers from Islamabad, the capital. Summer season in the district is hot and temperature may reach to 50 oC (122 oF) while, in winter temperature can be as low as 2-3 oC. The climate of the district is quite suitable for agriculture; wheat, rice, and sugarcane are its main field crops. However, Sargodha district is famous for citrus (Kinnow) production. The district Sargodha comprises of six tehsils i.e. Sargodha, Bhalwal, Sillanwali, Shahpur, Sahiwal and Kot Momin.
2.3.1.1.2 Multan
Multan is famous for saints and mango. There are many shrines of saints in the district. Many crops are grown in Multan but the only crop linked with its name is mango. Flat and alluvial plains are best combination for cultivation of fruit crops.
The canal system running in Multan brings soil from distant places and makes the land more fertile.
Multan has very harsh climate in summer and the temperature exceeds even
52 oC (126 oF) whereas, in extreme winter, the minimum recorded temperature is 44
approximately −1 oC (30 oF). Average rainfall is about 186 mm. Multan is also popular for its dust storms.
Basically, Pakistan is divided into ten agro-ecological zones depending upon climate, land utilization and availability of water (Fig.2.1). Maximum fruits and other field crops in Pakistan are cultivated in northern irrigated plan (Zone
IVa) among other agro-ecological zones. Following are the characteristics of Zone
IVa.
2.3.1.1.3 Northern irrigated plain (Zone IVa)
Northern Irrigated Plainis mostly characterized as flood plain with semi- arid to arid climate. The eastern side receives more average per annum rainfall (300 to 500 mm) than southwest (200 to 300 mm). The soils range from sandy, loam- clay to loam. The canal system in the northern part helps in cultivation of irrigated crops like wheat, rice, sugar cane and millets. The south is suitable for cotton, sugar cane, maize, citrus and mangoes (PARC, 1998).
Survey for the current study was planned in such a way that the areas which were best known for the target host plants must be visited. The survey was conducted in the citrus, mango and guava orchards for disease estimation as well as sampling. For citrus the survey was conducted in tehsils of district Sargodha. In
Sargodha district a total of 88 locations with three orchards from each location were surveyed (Table 2.1). In each orchard randomly 5 out of 40 trees were observed. Hence a total of 264 citrus orchards were surveyed and a total of 1320 trees were observed for disease estimation and sampling. The orchards were surveyed using double diagonal method. Randomly five trees from an orchard were observed for recording the data. 45
For mango, the surveys were conducted in 5 districts of Punjab viz.
Khanewal, Multan, Muzaffar Garh, Rahim Yar Khan and Bahawalpur. In total 50 locations were visited, in each location 3 mango orchards were surveyed and in each orchard 5 out of 35 trees were observed. A total of 150 mango orchards were surveyedand 750 mango trees were observed (Table 2.2).For disease assessment in guava, surveys were done in disttrict Kasur,Sheikhupura and Nankana Sahib. In total 26 locations and in each locationthree orchards were surveyed. Similarly from each orchard, 5 trees out of 40 were observed for the disease assessment and sample collection. Thus a total of 90 orchards and 390 trees wereobserved (Table
2.3).
46
I: Indus Delta
II: Southern Irrigated
III (a & b) Sandy Desert
IV (a & b) Nothern Irrigated Plains
V Barani Lands
VI Wet Mountains
VII Northern Dry Mountains
VIII Western Dry Mountains
IX Dry Western Plateau
X Suleman Piedmont
Fig. 2.1: Agro-ecological zones of Pakistan 47
Table 2.1: Areas surveyed for citrus decline assessment in district Sargodha.
S. No. Tehsils Localities
1 Bhalwal Chak No. 4 SB, 7 ML, 7SB, 7 ASB 8NB, 9
NB, Chak 9 Lokri, 10 NB, 13 NB, 18 NB, 22
NB, 23 NB, 26 NB
2 Sargodha Chak No. 24 SB, 27 SB, 28 SB, 30 NB, 48
NB, 53 SB, 56 NB, 90 NB, 91 NB, 93 SB, 94
BN, 95 NB, 95SB, 101 SB, 112 NB, 115 SB,
and 122 SB.
3 Sillanwanli Chak No. 118 NB, 119 SB, 120SB, 122 SB,
4123 SB, 124 SB, 127 SB, 127 NB,137 SB,
147 NB and 148 NB.
4 Shahpur Malakwal, Wadhi, kot Maghrib, Shahpur
Saddar, Noor Kallu, Kandaan Kalan,
Jhavarian, Hussain Shah, Kudyana, Allahdad
Wala, Chachar Sharf, Chak 128 NB
5 Sahiwal Kot Pehalwan, Biral Sharif, Nawabpur, Vijh,
Nehang, Chohal, Tirkhanwala, Haveli
Majuka, Pindi Wala, Sial Sharif, Sangoraka,
Farooka, Sial Dholka, Muhammad Wala,
Jahane Wala, Dherowal, Chatror and Radhan 48
S. No. Tehsils Localities
6 Kot Momin Chak No. 9 SB, 19 SB, 20 SB, 21 SB, 65 SB,
66 SB, Rawan, Dera Thoye Wala, Ghulapur
Bangla, Jalla Makhdum, Takht Hazara,
Naseerpur Kalan, Mateela, Dodha and Midh
Road
49
Fig. 2. 2: Areas surveyed for citrus decline in district Sargodha, Punjab, Pakistan
50
Table 2.2: Areas surveyed for mango decline assessment in various districts of
Punjab.
S. No. District Tehsils Localities
1 Khanewal Kabirwala Qadirpur Rawan, 5 Kassi,
Solgi, Matti Tal, Abbas Pur,
8 Kassi, Maula Pur, Bilawal
Pur, Basti Toheed Nagar and
Hassan Pur
2 Multan Multan Qasim Bela, Nandla, Chah
Nizam Wala, and Basti Band
Bosan.
Shujabad Basti Khokhran, Shahpur
Ubbha, Abbas Pura
Jalalpur Pirwala Ghazipur, Basti Malkani,
Manik Wali
3 Muzaffar Garh Muzaffar Garh Makhan Bela, Rohillanwali,
Shah Jamali
Kot Addu Ali Wala, Musay Wala,
Shuhrat Wala, Basti Drigh
Alipur Muradpur Pull, Basti 51
S. No. District Tehsils Localities
Nukray, Basti Jat Lashari
4 Rahim Yar Khan Rahim Yar Khan Taranda Muhammad Panah,
Wahi Shah Muhammad,
Mianwali Qureshian
Sadiqabad Ahmad Pur Lumma
Khan Pur Chak 2P, Chak 3 P, Bagh o
Bahar,
Liaqat Pur Chak 22 A, Chak 23 A,
Islam Nagar
5 Bahawalpur Bahawalpur Khanqah Sharif, Chak 13
BC, Chak 23 BC, Nowshera,
Munshi Wala
52
Fig. 2. 3: Areas surveyed for mango decline in district Khanewal, Punjab, Pakistan
53
Fig. 2. 4: Areas surveyed for mango decline in district Multan, Punjab, Pakistan
54
Fig. 2. 5: Areas surveyed for mango decline in district Muzaffar Garh, Punjab,
Pakistan
55
Fig. 2. 6: Areas surveyed for mango decline in district R. Y. Khan, Punjab,
Pakistan
56
Table 2.3: Areas surveyed for guava decline assessment in various districts of
Punjab.
S. No. District Tehsils Localities
1 Kasur Kasur Qaisar Garh, Dolaywala,
Noor Shah Wali, New City
Kasur, Sadar Diwan
Chuniyan Khara, Kot Gurdas Wala
Pattoki Gehlan Pathak, Rukan Pura,
Sehjowal
3 Sheikhupura Ferozewala Faizpur interchange, Adda
Thabal, Noor Shah, Burj
Attari, Saggian Khurd,
Thikriwala
Sharaqpur Sharaqpur, Ghareebabad,
Sukhanwala, Kot Mahmood
4 Nankana Sahib Nankana Sahib Jalal Nou, Giller Wala,
Magtan Wala, Adda Pul
Torian, Chachkay Gill,
Mirza Pur, Mandi Faziabad
and Mor Khunda
57
Fig. 2. 7: Areas surveyed for guava decline in district Kasur, Punjab, Pakistan
58
Fig. 2. 8: Areas surveyed for guava decline in district Sheikhupura, Punjab,
Pakistan
59
Fig. 2. 9: Areas surveyed for guava decline in district Nankana Sahib, Punjab,
Pakistan
60
Prevalence, incidence and severity data were recorded to calculate the disease index % age. Others parameters were also recorded which included tree age, tree height, variety, damage, symptom of the decline or sudden death, irrigation and fertilizer regimes etc.
2.3.2. Disease Incidence
The incidence of decline in citrus, mango and guava was calculated by the formula (Rehman et al., 2011 a).
Disease Incidence (%)= Number of declined plants× 100 Total number of plants observed
2.3.3. Disease Severity
Disease Severity of mango, citrus and guava decline was recorded using 0-5 visual rating scale(Kazmi et al., 2005). Where:
0=Healthy Plants; 1=1-10%decline; 2=11-20%; 3=21-30%, 4=31-50% 5=More than 50%.
2.3.4. Disease Index
Disease Index gives the exact status of the disease combining the disease incidence and severity (Kazmi et al., 2005).
Disease Index (%) = 0(n1)+1(n2)+2(n3)+3(n4)+4(n5)+5(n6)× 100 N 5
Where
n1: No. of trees in 0 rating
n2: No. of trees in 1 rating
n3: No. of trees in 2 rating 61
n4: No. of trees in 3 rating
n5: No. of trees in 4 rating
n6: No. of trees in 5 rating
N: Total Number of Trees
2.3.5. Symptomatology
Symptoms were carefully observed from each decline affected citrus, mango and guava trees. For current study, 50 trees each of citrus, mango and guava were selected and the %age of trees showing decline symptoms such as gummosis, bark splitting, die back, canker formation, stem bleeding etc was calculated.
2.4. RESULTS
2.4.1. Disease Assessment
As a result of survey for the disease assessment and observation taken, the disease incidence, severity and disease index was calculated as follows:
2.4.1.1. Citrus decline assessment
Disease incidence, severity and disease index were found to be variable in district Sargodha. Maximum mean disease incidence was recorded in tehsil
Sargodha (94.06%) followed by tehsil Shahpur (93.33%) and Sahiwal (73.33%), while it was the minimum in tehsil Sillanwali (35.73%). Similarly, mean disease severity was the maximum in tehsil Sargodha (1.47) and the minimum (0.64) wasobserved in Sillanwali. The rest of the tehsils has intermediate disease situation.
Likewise, maximum disease index was observed in tehsil Sargodha (29.41%) followed by tehsil Shahpur (26.75%) and Bhalwal (23.77%) On the other hand 62
minimum disease index was found in tehsil Sillanwali (10.36%) as shown in table
2.4.
In tehsil Bhalwal of Sargodha almost 54% of the locations had 100% disease incidence. Similarly, 61.5% locations fell into disease severity rating of "2" which was the maximum rating observed in any location of tehsil Bhalwal (Table
2.5).
In tehsil Kot Momin 27% of the locations showed 100% disease incidence while 80% locations had disease severity rating of "1", maximum rating observed in any location of tehsil Kot Momin. Disease index ranged from 12-28% as shown in Table 2.6.
In tehsil Sargodha, 71% of the locations observed had 100% disease incidence and in 47% locations maximum disease severity rating "2" was recorded.
Disease index ranged from 20-40% (Table 2.7). In tehsil Sahiwal, 61% locations observed had 100% disease incidence; 11% locations showed disease severity rating "2" (maximum). Disease index ranged from 12-32% as shown in Table 2.8.
In Shahpur tehsil 58% locations had 100% disease incidence and 25% locations showed maximum disease severity rating "2" while disease index ranged from 19-35% (Table 2.9). Similarly, in Sillanwali only one location had 100% disease incidence maximum disease severity rating was "2". Disease index however, ranged from 4-32% (Table 2.10)where growers are following integrated orchard management approach (Fateh et al., 2017).
Generally disease index of more than 15% of any disease at any location or any host is considered alarming. However, in district Sargodha overall disease index ranged from minimum 10.36 to maximum 29.41% which is a real panic for 63
the growers as well as entire citrus industry and requires the immediate attention of the scientists and other stakeholders. There is lack of value chain and supply chain studies and training of the growers‟ at large scale based on the best available integrated approaches. Nominal quarantine measures in the country has also aggravated the disease situation which renders the growers to either change their crops or they sell their properties to land mafia for housing societies. It is concluded that the situation of citrus decline is alarming in district Sargodha and almost every orchard faced the problem. The situation is alarming and warrants strict control strategies for its management. It is also emphasized that the farming community be trained in this regard.
2.4.1.2. Mango decline assessment
According to results shown in Table 2.11, maximum mean disease severity
2.70 was observed in district Bahawalpur followed by Khanewal (2.30), Multan
(2.11), Muzaffar Garh (2.00). Minimum disease severity (1.90) was recorded in
Rahim Yar Khan District.
Similarly maximum mean disease incidence (100%) was found in
Khanewal followed by Bahawalpur (99.30%), Muzaffar Garh (96.00%), Multan
(96.00%) and minimum disease incidence (90.00%) was observed in Rahim Yar
Khan.Disease index which gives the actual picture of the disease was found to be the maximum in Bahawalpur (53.30%) followed by Khanewal (46.50%), Rahim
Yar Khan (39.10%), Muzaffar Garh (37.30%) while minimum disease index 36.89 was found in Multan district (Table 2.11).
According to table 2.12, in tehsil Kabirwala of district Khanewal mean disease severity raged from 2-3 based on 0-5 rating scale. Maximum mean disease 64
Table 2.4: Mean citrus decline incidence, severity and disease index in different
tehsils of district Sargodha.
Sr. Tehsil Mean Mean Disease Disease No. Disease Incidence (%) Index (%) Severity (0-5) 1 Bhalwal 1.38 72.85 23.77 2 Kot Momin 0.80 64.87 15.80 3 Sargodha 1.47 94.06 29.41 4 Sahiwal 0.89 73.33 19.28 5 Shahpur 1.25 93.33 26.75 6 Sillanwali 0.64 35.73 10.36 Data are means of five replicates
Table 2.5: Mean citrus decline incidence, severity and disease index in different
locations of tehsil Bhalwal of district Sargodha.
Sr. Location Mean Mean Disease Disease No. Disease Incidence (%) Index (%) Severity (0-5) L Chak No. 4 SB 2 100 31 2 Chak No. 7 ML 2 100 32 3 Chak No. 7SB 2 100 40 4 Chak No. 7 ASB 2 100 37 5 Chak No. 8NB 2 100 33 6 Chak No. 9 NB 1 67 15 7 Chak No. Chak 9 Lokri 2 100 36 8 Chak No. 10 NB 0 0 0 9 Chak No. 13 NB 2 93 31 10 Chak No. 18 NB 2 100 37 11 Chak No. 22 NB 0 0 0 12 Chak No. 23 NB 1 87 17 13 Chak No. 26 NB 0 0 0 Data are means of five replicates
65
Table 2.6: Mean citrus decline incidence, severity and disease index in different
locations of tehsil Kot Momin of district Sargodha.
Sr. Location Mean Mean Disease Disease No. Disease Incidence (%) Index (%) Severity (0-5) 1 Chak No. 9 SB 0 0 0 2 19 SB 0 0 0 3 20 SB 1 60 12 4 21 SB 1 80 19 5 65 SB 1 73 16 6 66 SB 1 87 23 7 Rawan 0 0 0 8 Dera Thoye Wala 1 100 24 9 Ghulapur Bangla 1 100 29 10 Jalla Makhdum 1 80 16 11 Takht Hazara 1 60 12 12 Naseerpur Kalan 1 73 17 13 Mateela 1 100 25 14 Dodha 1 100 28 15 Midh Road 1 60 16 Data are means of five replicates
66
Table 2.7: Mean citrus decline incidence, severity and disease index in different
locations of tehsil Sargodha of district Sargodha.
Sr. Location Mean Mean Disease Disease No. Disease Incidence (%) Index (%) Severity (0-5) 1 Chak No. 24 SB 1 80 20 2 Chak No 27 SB 2 100 37 3 Chak No 28 SB 1 73 20 4 Chak No 30 NB 1 100 27 5 Chak No 48 NB 1 100 29 6 Chak No 53 SB 2 100 31 7 Chak No 56 NB 1 100 27 8 Chak No 90 NB 2 100 32 9 Chak No 91 NB 1 73 24 10 Chak No 93 SB 2 100 40 11 Chak No 94 NB 2 100 37 12 Chak No 95 NB 2 100 33 13 Chak No 95SB 2 100 32 14 Chak No 101 SB 1 73 24 15 Chak No 112 NB 1 100 29 16 Chak No 115 SB 2 100 31 17 Chak No 122 SB 1 100 27 Data are means of five replicates
67
Table 2.8: Mean citrus decline incidence, severity and disease index in different
locations of tehsil Sahiwal of district Sargodha.
Sr. Location Mean Mean Disease Disease No. Disease Incidence (%) Index (%) Severity (0-5) 1 Kot Pehalwan 1 100 28 2 Biral Sharif 1 93 25 3 Nawabpur 1 100 24 4 Vijh 1 100 28 5 Nehang 0 0 0 6 Chohal 1 100 23 7 Tirkhanwala 0 0 0 8 Haveli Majuka 2 100 32 9 Pindi Wala 1 100 24 10 Sial Sharif 2 100 31 11 Sangoraka 1 60 12 12 Farooka 1 67 16 13 Sial Dholka 1 100 23 14 Muhammad Wala 1 100 24 15 Jahane Wala 0 0 0 16 Dherowal 1 100 28 17 Chatror 1 100 29 18 Radhan 0 0 0 Data are means of five replicates
68
Table 2.9: Mean citrus decline incidence, severity and disease index in different
locations of tehsil Shahpur of district Sargodha.
Sr. Location Mean Mean Disease Disease No. Disease Incidence (%) Index (%) Severity (0-5) 1 Malakwal 1 67 19 2 Wadhi 2 87 35 3 Kot Maghrib 1 93 20 4 Shahpur Saddar 1 100 27 5 Noor Kallu 1 100 27 6 Kandaan Kalan 2 100 31 7 Jhavarian 1 100 28 8 Hussain Shah 1 80 25 9 Kudyana 1 93 23 10 Allahdad Wala 1 100 25 11 Chachar Sharf 1 100 28 12 Chak 128 NB 2 100 33 Data are means of five replicates
Table 2.10: Mean citrus decline incidence, severity and disease index in different
locations of tehsil Sillanwali of district Sargodha.
Sr. No. Location Mean Disease Mean Disease Disease Severity (0-5) Incidence (%) Index (%)
1 Chak No. 118 NB 1 33 15 2 Chak No. 119 SB 1 73 24 3 Chak No. 120SB 0 0 0 4 Chak No. 122 NB 1 53 12 5 Chak No. 123 SB 2 100 32 6 Chak No. 124 SB 0 0 0 7 Chak No. 127 SB 0 20 4 8 Chak No. 127 NB 1 67 15 9 Chak No. 137 SB 0 0 0 10 Chak No. 147 NB 0 0 0 11 Chak No. 148 NB 1 47 12 Data are means of five replicates 69
severity "3"was recorded in Matti Tal, Bilawal Pur and Basti Toheed Nagar followed by disease severity "2" in Qadirpur Rawan, 5 Kassi, Solgi, Abbas Pur, 8
Kassi, Maula Pur and Hassan Pur. Mean disease incidence observed was 100% in all orchards observed in all locations. However, maximum disease index 65% was observed in Basti Toheed Nagar followed by 57% in Bilawal Pur, 51% in Matti
Tal, 49% in Maula Pur, 47% in 8 Kassi, 41% in Solgi, 40% in Abbas Pur, 39% in
Qadirpur Rawan and 5 Kassi and minimum 37% in Hassan Pur.
Survey condcuted in 3 tehsils of Multan showed mean disease severity ranging from 1-3. Maximum mean disease severity "3"was observed in Chah
Nizam Wala (Tehsil & district Multan) and Ghazi Pur (Tehsil Jalalpur Pirwala, district Multan) followed by disease severity "2" in Nandla, Basti Band Bosan
(Tehsil Multan); Basti Khokhran, Shahpur Ubbha, Abbas Pura (Tehsil Shujabad) and Manik Wali ( Tehsil Jalal Pur Pirwala). Minimum mean disease severity "1" was observed in Qasim Bela of Multan. Mean disease incidence was 100% in all orchards except Qasim Bela and Nandla in tehsil Multan where it was 67%.
Disease index was maximum in 52% in Chah Nizam Wala (Multan) and Ghazipur
(Jalalpur Pirwala) followed by 40% in Shahpur Ubbha and Abbas Pura (Shujabad),
39% in Manik Wali (Jalalpur Pirwala), 33% Bast Band Bosan (Multan), 32% Basti
Khokhran ( Shujabad), 31% Nandla (Multan) and minimum 13% in Qasim Bela of tehsil Multan (Table 2.13).
In Muzaffar Garh district, 3 tehsils were covered i.e. Muzaffar Garh, Kot
Addu and Ali Pur. Mean disease severity ranged from 1-3. Maximum mean disease severity "3" was recorded in Basti Drigh of Muzaffar Garh tehsil and Basti Nukray of Alipur tehsil followed by disease severity "2" in Makhan Bela, Rohillanwali, 70
Shah Jamali (Tehsil Muzaffar Garh), Shuhrat Wala (Kot Addu), Muradpur Pull and
Basti Jat Lashri (Ali Pur). Minimum mean disease severity "1" was observed in Ali
Wala and Musay Wala of tehsil Kot Addu, District Muzaffar Garh. Mean disease incidence 100% was observed in Rohillanwali, Shah Jamali (Muzaffar Garh); Ali
Wala and Shuhrat Wala, Basti Drigh (Kot Addu) and Murad Pur Pull, Basti Nukray and Basti Jatt Lashri of tehsil Ali Pur. Maximum disease index (52%) was calculated in Basti Drigh of Kot Addu tehsil and Basti Nukray of Ali Pur tehsil followed by 44% in Makhan Bela and Shah Jamali (Muzaffar Garh); 41% Basti Jat
Lashari (Ali Pur), 40% Shuhratt Wala (Kot Addu); 31% Muradpur Pull (Ali Pur);
29% Ali Wala (Kot Addu), 27 % Rohillanwali (Muzaffar Garh) and minimum 13%
Musay walla (Kot Addu) (Table 2.14).
In district Rahim Yar Khan 4 tehsils i.e. Rahim Yar Khan, Sadiqabad, Khan
Pur and Liqat Pur were surveyed. Maximum mean disease severity "3" was observed in Wahi Shah Muhammad of tehsil Rahim Yar Khan only followed by disease severity "2" in all locations. Disease incidence was 100% in all locationssurveyed. However, maximum disease index (52%) was found in Wahi
Shah Muhammad (Rahim Yar Khan) followed by 49% in Chak 23 A (Liaqat Pur);
48% Chak 2P (Khan Pur). and Islam Nagar (Liaqat Pur); minimum 37 % in
Taranda Muhammad Panah & Mianwali Qureshian of tehsil Rahim Yar Khan
(Table 2.15).
Tehsil Bahawalur and Ahmedpur East were surveyed in the district of
Bahawalpur. Mean disease severity ranged from 2-4 based on 0-5 rating scale.
Maximum mean disease severity "4" was recorded in Chak 23 BC of tehsil
Bahawalpur followed by "3" in Khanqah Sharif, Chak 13 BC, Munshi Wala (Baha- 71
Table 2.11: Mean mango decline incidence, severity and disease index in different
districts of Punjab province.
Sr. No. District Mean Disease Mean Disease Disease Severity Incidence (%) Index (%) (0-5) 1 Khanewal 2.30 100 46.50 2 Multan 2.11 92.67 36.89 3 Muzaffar Garh 2.00 96.00 37.30 4 Rahim Yar Khan 1.90 90.00 39.10 5 Bahawalpur 2.70 99.30 53.30 Data are means of fivereplicates
Table 2.12: Mean mango decline incidence, severity and disease index in different
location of district Khanewal.
Sr. No. Tehsil Location Mean Disease Mean Disease Disease Severity (0-5) Incidence (%) Index (%)
1 Kabirwala Qadirpur Severity2 (0-5) 100 39 5 Kassi 2 100 39 RawanSolgi 2 100 41 Matti Tal 3 100 51 Abbas Pur 2 100 40 8 Kassi 2 100 47 Maula Pur 2 100 49 Bilawal Pur 3 100 57 Basti 3 100 65 Hassan Pur 2 100 37 Toheed
Data are means of fivereplicates Nagar and
72
Walpur) and Mehrab Wala and Basti Johnan (Ahmedpur East). Minimum mean disease intensity "2" was recorded in Nowshera (Bahawalpur) and Channi Goth,
Muhabbat Pur and Basti Khokhran of tehsil Ahmedpur East. Mean disease incidence was 100% in all locations except in Khanqah Sharif (Bahawalpur) which was 93%. Maximum disease index 76% was observed in Chak 23 BC
(Bahawalpur) followed by 61% in Basti Johnan and it was minimum in Channi
Goth as shown in Table 2.16.
2.4.1.3 Guava decline assessment
During the decline assessment in guava, results showed that among three guava growing districts, the maximum severity was recorded in district
Sheikhupura (0.90) followed by district Kasur (0.78), while, the minimum severitywas observed in district Nankana Sahib (0.57).
Maximum guava decline incidence was found in district Kasur (62.89%) followed by Sheikhpura (61.30%) whereas it was minimum in district Nankana
Sahib (56.29%).
However, a maximum disease index of 18% was recorded in district Kasur followed by Sheikhpura but the minimum disease index of 14% was observed in district Nankana Sahib (Table 2.17).
73
Table 2.13: Mean mango decline incidence, severity and disease index in different
locations of district Multan.
Sr. No. Tehsil Location Mean Disease Mean Disease Disease
Severity (0-5) Incidence (%) Index 1 Multan Qasim Bela 1 67 13 Nandla 2 67 31(%) Chah 3Severity (0-5) 100 52 Nizam Basti Band 2 100 33 Wala Bosan 2 Shujabad Basti 2 100 32
ShahpurKhokhran 2 100 40 Abbas Pura 2 100 40 3 Jalalpur GhazipurUbbha 3 100 52 Manik Wali 2 100 39 Data are meansPirwala of five replicates
74
Table 2.14: Mean mango decline incidence, severity and disease index in different
locations of district Muzaffar Garh.
Sr. No. Tehsil Location Mean Disease Mean Disease Disease
Severity (0-5) Incidence (%) Index 1 Muzaffar Makhan 2 93 44 Rohillanwal 2 100 27(%) Garh ShahBela Jamali 2 100 44 Severity (0-5) 2 Kot Addu Alii Wala 1 100 29 Musay 1 67 13
ShuhratWala 2 100 40
BastiWala Drigh 3 100 52 3 Alipur Muradpur 2 100 31 Basti 3 100 52 BastiPull Jat 2 100 41 Nukray Data are means of five replicatesLashari
Table 2.15: Mean mango decline incidence, severity and disease index in different
locations of district Rahim Yar Khan.
Sr. Tehsil Location Mean Disease Mean Disease
No. Severity (0-5) Disease Index (%)
1 Rahim Yar Taranda 2 100Incidence 32 Wahi Shah 3 100 52 Khan MianwaliMuhammad 2 100(%) 32 Muhammad 2 Sadiqabad AhmadQureshianPanah Pur 2 100 41 3 Khan Pur Chak 2P 2 100 48 ChakLumma 3 P 2 100 44 4 Liaqat Pur Chak 22 A 2 100 45 Chak 23 A 2 100 49 Islam Nagar 2 100 48 Data are means of five replicates 75
Table 2.16: Mean mango decline incidence, severity and disease index in different
locations of district Bahawalpur.
Sr. No. Tehsil Location Mean Disease Mean Disease
Severity (0-5) Disease Index (%)
1 Bahawalpur Khanqah 3 93Incidence 53 Chak 13 BC 3 100 52 ChakSharif 23 BC 4 100(%) 76 Nowshera 2 100 49 Munshi 3 100 57
2 Ahmed Pur WalaChanni 2 100 32
East MehrabGoth 3 100 60
MuhabbatWala 2 100 48
BastiPur 2 100 45
BastiKhokhran, 3 100 61
Johnan Data are mean of five replicates.
76
In District Kasur mean disease severity of guava decline ranged from 0-1 based on 0-5 rating scale. Maximum mean disease severity "1" was recorded in all the villages except Saddar Diwan (Kasur) and Kot Gurdas Wala (Chuniyan).
Maximum mean disease incidence of 93% was recorded in Khara of tehsil
Chuniyan followed by Gehlan Phatak and Sehjowal of Pattoki tehsil (73%), while, the minimum was observed in Dolaywala (53%). Maximum disease index was observed in Sehjowal (24%) followed by Khara and Gehlan Phatak (23%). On the other hand minimum disease index (17%) was found in Dolaywala (Kasur) (Table
2.18).
In Sheikhpura district 2 tehsils were surveyed i.e. Ferozewala and
Sharaqpur. Maximum mean disease severity "2" was observed only in Faizpur interchange. Disease severity " 1" was in all the locations except in Sharaqpur and
Sukhanwala (Tehsil Sharaqpur). Maximum mean disease incidence of 93% was observed in Faizpur interchange followed by 80% in Noor Shah, while, the minimum incidence was recorded in both Ghareebabad and Kot Mahmood.
Maximum disease index 33% was recorded in Faizpur Interchange followed by
28% in Burj Attariand it was minimum (12%) in Ghareebabad (Table 2.19).
In tehsil and district Nankana mean disease severity of "1" was observed in more than 50% locations. Similarly,disease incidence 80% in Adda Pull Torian followed by 67% in Jalal Nou. Maximum disease index in district Nankana was observed 27% in Adda Pull Torian followed by Chackay Gill (19%), Jalal Nou
(15%) and minimum 12 % in Mirzapur (Table 2.20).
2.4.2. Symptomatology 77
The symptoms development percentageswere calculated by randomly selecting 50 trees of each citrus, mango and guava. According to Table 2.21, recorded data showed that dieback was maximum (42%) in guava followed by 38% in citrus and minimum (28%) in mango trees. Bark splitting was shown to be maximum by mango (38%), while, in citrus and guava bark splitting trees were
30% and 16% respectively. Maximum gummosis was recorded in 48%mango trees, followed by 42% in citrus and minimum 32% in guava. Cankers were observed in
36% citrus trees (maximum), while in mango and guava it was 20% and 10% respectively. Thirty percent (30%) mango trees showed stem bleeding followed by
28% in citrus and 8% in guava trees. Maximum mortality (8%) was observed in mango, followed by (4%) guava and 3% citrus trees (Table 4.5). It was noted that the similar symptoms observed in citrus, mango and guava included twigs dieback
(Fig. 2.10), bark splitting and gummosis (Fig.2.11, 2.14and 2.17), canker development (Fig. 2.12, 2.2.15 (b) and 2.18. Finally, the mortality of decline affected trees occur (Fig. 2.13, 2.16 and 2.19).
The results show that all citrus, mango and guava tree showed somewhat similar symptoms with greater or lesser extent. These symptoms may appear either due to similar predisposing factors or due to the common pathogens. However, bark splitting in citrus, mango and guava trees happen differently, this is may be due to their wood structure, physiology or the varietal character. Moreover, sudden death is found less in citrus and guava as compared to mango. Dieback symptoms have been most common among target commodities. Similarly canker development can also be seen very commonly in all cases. Leaf drooping and drying but remaining attached to trees was a common symptom in sudden death. 78
Table 2.17:Mean guava decline incidence, severity and disease index in different
districts of Punjab province.
Sr. District Mean Mean Disease Index
No. Disease Disease (%)
1 Kasur 0.78Severity 62.89Incidence 18.00 2 Sheikhupura 0.90 61.30 17.40 3 Nankana Sahib 0.57 (0-5) 56.29(%) 14.00
Data are mean of five replicates
Table 2.18: Mean guava decline incidence, severity and disease index in different
locations of district Kasur.
Sr. Tehsil Location Mean Mean Disease
No. Disease Disease Index (%)
1 Kasur Dolaywala 1Severity 53Incidence 17 Noor Shah 1 67 20 (0-5) (%) NewWali City 1 67 19
Sadar Diwan 0 47 9 2 Chuniyan KharaKasur 1 93 23 Severity (0- Kot Gurdas 5)0 33 8
3 Pattoki GehlanWala 1 73 23
PathakRukan Pura 1 60 19
Sehjowal 1 73 24
Data are mean of five replicates
79
Table 2.19: Mean guava decline incidence, severity and disease index in different
location of district Sheikhupura.
Sr. Tehsil Location Mean Mean Disease
No. Disease Disease Index (%)
1 Ferozewala Faizpur 2Severity 93Incidence 33 Adda Thabal 1 67 16 interchange, (0-5) (%) Noor Shah 1 80 17
Burj Attari 1 67 28 Saggian Severity1 (0- 67 19
KhurdThikriwala 5)1 60 17
2 Sharaqpur Sharaqpur 0 33 8
Ghareebabad 1 53 12
Sukhanwala 0 40 9
Kot 1 53 15
Data are mean of five replicatesMahmood
80
Table 2.20: Mean guava decline, severity and disease index in different location of
district Nankana Sahib.
Sr. Tehsil Location Mean Mean Disease
No. Disease Disease Index (%)
1 Nankana Jalal Nou 1Severity 67Incidence 15
Sahib Giller Wala 0(0 -5) 47(%) 9 Magtan Wala 0 40 8 Adda Pul 1 80 27 Severity (0- Torian Chachkay 5)1 60 19 Gill Mirza Pur 1 60 12
Mandi 0 40 8
Data are mean of five replicatesFaziabad
81
Fig.2.10: Twig dieback in citrus.
Fig.2.11: Bark Splitting and gummosis in citrus.
82
Fig.2.12: Canker development in citrus.
Fig.2.13:Citrus tree mortality. 83
Fig.2.14: Bark splitting and oozing of thick dark brown liquid in Mango.
Fig. 2.15 (a) Fig. 2.15 (b)
Fig.2.15 (a) and (b): Gummosis and stem canker in mango decline affected tree.
84
Fig.2.16: Mango Sudden death affected dead tree (MSD).
Fig. 2.17: Bark splitting and gummosis in decline affected guava tree. 85
Fig. 2.18: Tree canker in decline affected guava tree.
Fig. 2.19: Decline affected dead guava tree.
86
Table 2.21: Percentage of citrus, mango and guva trees showing various decline
symptoms.
Orchards Die- Bark Gummosis Canker Stem Complete back splitting (% trees) formation bleeding Mortality (% trees) (% trees) (% trees) (% trees)
19/50 15/50 21/50 18/50 14/50 3/50 Citrus (38%) (30%) (42%) (36%) (28%) (6%)
14/50 19/50 24/50 10/50 15/50 4/50 Mango (28%) (38%) (48%) (20%) (30%) (8%)
21/50 8/50 16/50 5/50 4/50 2/50 Guava (42%) (16%) (32%) (10%) (8%) (4%)
Data are mean of 50 replicates
87
2.5. DISCUSSION
The overall results indicate disease havoc in citrus growing areas of district Sargodha which is due to a number of biotic and abiotic factors. Most of the scientists thought that it was CTV causing et al., 2009 and Arif et al., 2015). Others thought that phytoplasma and bacteria are involved in citrus decline (Burney et al.,
2007). Some considered bacteria e.g. Xanthomonas compestris as the cause of citrus decline. Fungal involvement in citrus decline was studied in Oman, Iran and
Pakistan. A list of fungi has been reported to cause decline symptoms. e.g.
Lasiodiplodia hormozganensis, L. theobromae, Fusarium solani, Phytophthora sp.,
Neoscytalidium dimidiatum and Nattrassia mangiferaebeing the most common
(Safdar etal., 2010).
Among abiotic stresses there are two categories which are related to soil i.e. salinity, nutrient toxicity/ deficiency, compaction/ hardpan in subsurface and other is irrigation problem including water logging and water stress (Ali et al., 2014).
The human manipulations regarding orchard management as well as intercropping are aggravating decline problem in existing citrus growing areas. Most of the orchards in Sargodha, Bhalwal and Shahpur had old citrus plantations with intercropping of wheat, berseem and barley. Removal of deadwood and pruning are not commonly practicedin these orchards which haveresulted in profused branching of trees. However, better situation was found in terms of orchard management in
Kot Momin and Sillanwali tehsils.
In mango areas, maximum decline disease index of 53.30% was recorded in district Bahawalpur. During survey the large overlapping trees have been observed in this district. Most of the areas surveyed had more than 100 years old plantations. 88
The age of trees have been reported to have significant relationship with decline symptoms (Khaskheli et al., 2011). The growers were not even aware of the disease but they thought that senescens had been resulting in such symptoms of the trees. Secondly mango orchards were not given importance by the growers rather they were taking care of intercropped plants. The large farms were owned by the absentee landlords who stayed abroad and left orchard management on their attendants. These attendants strongly believed in traditional mango cultivation and showed harshness towards the adoption of innovative technologies.
In case of guava, maximum disease index was found in Kasur (18%) followed by Sheikhupura (17.40%) and minimum 14% in Tehsil Nankana. Kasur being the nursery industry area is looking for guava trees in high density for sale rather than better management to earn from the fruit. Most of the trees were not grown in proper rows, rather overlapping with each other in such a way that all the operations like sprays, pruning and machine operations were difficult. Improper cuttings and non treatment of woundsresulted in guva decline. However, proper guava cultivation has been observed in Sheikhupura with intercropping of wheat and berseem. Ploughing inside the orchards increased tree infection by making injuries. Nankana is comparatively low in disease as most of the platations were with proper planning and better management strategy. The main reason for low severity in Nankana Sahib District may bedue to a new cultivaing area for guava; the trees are young and are planted in a systematic way.
89
Chapter 3
PREVALENCE OF NATTRASSIA SP. FROM CITRUS, MANGO AND GUAVA ORCHARDS ALONG WITH OTHER DECLINE CAUSING FUNGI 3.1. INTRODUCTION
Tree decline in citrus, mango and guava has been a hot issue in Pakistan and some of the countries around the globe. Different researchers around the world have reported different causes of these declining trees. Mostly described terms for the decline factors are predisposing, inciting and contributing factors (Malik et al.,
2004; Safdar et al., 2010 and Safdar et al., 2015).
Predisposing factors are long term factors that change slowly such as soil, site and climate. These affect tree's ability to respond to any kind of injury or disease inducing agents. In case of citrus, mango and guava decline all of these factors have a key role. The changing climate especially prevailing drought conditions have tremendously affetced citrus, mango and guava cultivation as well as other fruit, forest and ornamental trees. Similarly, sites also play important role as the plantation near the roads or canals are mostly decline affected either because of hard soil pans, road or canal widening and other human interventions which cause the root injury of the trees and result in the decline symptoms. Saline and saturated soils are other predisposing factors which create hindrance in the orchard management. Salinization of soil and water is an important factor for increased desertification in arid and semi-arid regions of the world (Szabolcs, 1992). About
6.3 million hectares of arable land in Pakistan is affected to varying degrees of salinity/sodicity.
64 90
Inciting factors are of short duration and appear in biological or physiological form. They generally produce dieback symptoms or we can say they are just starter of the disease. These might be twig dieback causing fungi, insects, phytoplasma, late spring frost, drought and salt spray (Burney et al., 2007).
The role of contributing factors starts after inciting factors which further weaken the plants and ultimately moratlity occurs. These include pathogenic fungi, vectors (in case of decline the bark beetle), canker causing fungi, bacteria, viruses and phytoplasma etc (Kazmi et al., 2005).
The most important pathogens worked out among other contributing biotic and abiotic factors causing tree decline are the fungi. Maximum fungi have been isolated from the roots of decline affected citrus trees. The isolated fungi were
Nattrassia mangiferae, Fusarium sp., Alternaria sp. and Aspergillus sp. from the area of Sargodha, Bhalwal, Kot Momin, Sillanwali and Toba Tek Singh areas of the Punjab province (Khan et al., 2012; Ali et al., 2014). Several fungi isolated from decline affected mango trees include Ceratocystis fimbriata, Lasiodiplodia theobromae, Fusarium sp., Nattrassia mangiferae, Phomopsis sp. (Kazmi et al.,
2005; Fateh et al., 2006 and Masood et al., 2010). Fungi associated with guava decline include Fusarium sp., Nattrassia mangiferae, Macrophomina sp.,
Cephalosporium sp., Alternaria sp., Pythium sp., Verticillium sp., Trichthecium sp.
(Mirzae, 2003; Miskita et al., 2005).
It is hypothesized that sometimes these fungi individually or sometimes with the interaction of more than one fungi result in the appearance of decline symptoms. These different fungi are found in different profiles or parts of the decline affected trees. The fungus N. mangiferae have a wide host arrange around 91
the globe and has covered field crops, ornamental, forest and fruit trees. However, in Pakistan, the fungus was not studied thoroughly on systematic basis. Hence, the present study was conducted to determine the frequency of Nattrassia mangiferaealong with other mycoflora associated with decline of citrus, mango and guava and to find out common mycoflora in different plant parts of the affected trees so that a common management may be devised for decline of all fruit crops.
3.2. REVIEW OF LITERATURE
Lonsdale (1992) reported that Nattrassia mangiferae was found to be an important pathogen of mango in South Africa. It was found to be the cause of blossom blight. Initial symptoms of the disease were sudden wilt followed by rapid dying and drying out of inflorescences. In the later stages of the disease, side axes and individual flowers dropped, fruitlets shriveling up and turned black and also dropped. Other fungi such as Alternaria alternata and Colletotrichum gloeosporioides did not cause blight, but instead, induced small, dark necrotic spots
(lesions) on the inflorescences.
In North America, eucalyptus dieback for the first time was reported in
1994 caused by the fungus N. magniferae. Dieback symptoms were observed in branches; rifting between the phloem and the xylem. During summer cankers and exuding gum were also observed on Eucalyptus camaldulensis Dehnh. trees in southwestern Arizona. N. mangiferae was the dominant fungus which has been consistently isolated from the affected branches (Michael, 1994).
In south-eastern Iran, the fungus N. mangiferae was reported from guava and Ficus religiosa, causing branch die-back. The pathogenicity was confirmed through Koch's postulates. This pathogen repported for the first time in 2000 in 92
Iran and has been a threat to Irani citrus and guava industry since then (Mirzaee et al., 2003).
Decline of Pacific Madrone has been a popular disease caused by N. mangiferae inducing cankers on stem and blight of shoots. Due to attack by N. mangiferae trees were weakened and opportunistic fungi like Fusicoccum aesculii nvaded stressed trees. These two pathogens were also reported on declining
Madrone trees in Washington, USA but N. mangiferae was the most prominent and primary pathogen (Elliott and Robert, 2002).
Further studies on the fungus N. mangiferae causing canker of Pacific madrone were based on morphological and molecular methods. The asexual spores confirm the pathogen, however, sequencing of the ITS region of the ribosomal rDNA of sexual state had linkages with genus Botryosphaeria. The fungus showed resemblance to Fusicoccum anamorphs, closely related Botryosphaeria species and has a similar pathology (Elliott and Robert, 2003).
Msikita (2005) isolated the fungus N. mangiferae from cassava causing root rot in Bénin and West Africa. The pathogenicity was confirmed on different cultivars of cassava, showing fungal lesions, while control plants remained symptom free. Other pathogens causing root rot included Macrophomina phaseolina, Fusarium sp. B. theobromae and Pythium sp.
David et al. (2005) reported that pacific Madrone had been facing decline for thirty years. It was reported that fungus causing decline has been present in western North America since 1968. Prior to this research, plant pathologists had misidentified the causal fungus, which is now known to be N. mangiferae. 93
Jayasinghe and Silva (2007) for the first time reported N. mangiferae causing foot canker of Hevea seedlings. One of the predisposing factors for the foot canker was sun-scorch and hence it must be considered in other declining trees as well where N. mangiferae is isolated.
3.3.MATERIALS AND METHODS
3.3.1. Sampling Methodology
During survey of citrus mango and guava orchards, samples were taken from the twigs, branches, stem at collar region, bark and the roots of decline affected trees. The samples were taken from randomly selected villages keeping in mind the maxiumum orchards present in the areas. For citrus samples were collected from samples 26 NB, 8NB and 10NB from tehsil Bhalwal of district
Sargodha. Similarly in tehsil and district Sargodha, samples were taken from 56
NB, 112 NB and 27 SB villages.
In mango growing areas the samples for isolation were taken from Nandla,
Chah Nizam Wala and Basti Band Bosan in district Multan while, in district Rahim
Yar Khan samples were taken from villages of Chak 22A (tehsil Liaqatpur), Chak
2P (tehsil Khan Pur) and Mianwali Qureshian.
To know the fungal frequencies in guava decline orchards, the samples were taken from Sehjowal, tehsil Pattoki of district Kasur, Faizpur (tehsil
Ferozewala of district Sheikhupura) and Adda Pul Torian(tehsil and district
Nankana Sahib).
The samples were used for the isolation, identification and further pathogenicity studies. The sample pieces were cut with sterilized sharp tool and kept in paper bags which after proper labeling were placed in the shopping bags to 94
avoid moisture loss.
3.3.2. Isolation and Identification of Pathogens
All the diseased samples of mango, citrus and guava were brought to
Mango Pathological Lab. at National Agricultural Research Centre, Islamabad and the isolations were made aseptically in the laminar flow chamber placed in isolation room. All glassware and media were sterilized by autoclaving at 121oC at
15 Psi for 20 min. For the isolation of fungi Potato Dextrose Agar (PDA), Malt
Extract Agar (MEA) and Stem Decoction Glucose Agar (SDGA) media were used.
Recipe of the media used is given below.
3.3.2.1.Potato dextrose agar medium (PDA)
For making Potato Dextrose Agar (PDA) medium a readymade mixture of the chemical company BDH was used
Potato Dextrose Agar = 39 g
Sterilized Distilled water = to make volume up to 1000 ml
Medium was made homogeneous by heating in a microwave oven for 3 minutes in half filled 1000 ml flasks, air tightened with cotton plug and later sterilized by autoclaving at 121oC at 15 Psi for 20 minutes.
3.3.2.2.Malt extract agar (MEA)
This medium was prepared by mixing following ingredients:
Malt Extract= 20 g
Agar= 20 g
Distilled water= to make volume up to 1000 ml
3.3.2.3. Stem decoction glucose agar (SDGA)
Citrus/mango/guava stem pieces = 200 g 95
Agar= 20 g
Glucose=20 g
Distilled water= to make volume up to 1000 ml
The respective stem pieces of citrus, mango and guava were boiled in 1000 ml water for 10 min. cooled down and then again boiled so that half of the volume is left. Concentrated water sieved through muslin cloth in beaker and more water was added to make the volume up to 1 L before adding other ingredients.
3.3.2.4. Sterilization
The media were sterilized in autoclave at 121oC and 15 PSi. All glassware including flasks, petriplates and other inoculation equipment was also sterilized in the autoclave.
3.3.2.5. Isolation from infected tissues
All the infected tissue samples were cut into small pieces and disinfested by rinsing into 1 % sodium hypochlorite for 1 min. followed by washing thrice with sterilized distilled water. The washed pieces were dried on filter papers to remove excessive moisture and aseptically placed in the 90 mm petridishes containing either PDA or MEA medium. The plates were wrapped with parafilm to avoid moisture loss and contamination and incubated 25oC ±.
3.3.3. Microscopy
Slides were prepared by taking a minute portion of fungal colony from the growing margin with the help of inoculating needle and identified by viewing under the Labomed Florescent Microscope made in USA microscope,using the magnifications of X100, X200, X400 and X1000. Isolated fungi were identified on the basis of their colony characteristics, conidial morphology, ascospores, pycnidia, 96
pericthecia etc. The identification was based on key used by Fateh et al., 2010 and
Domsch et al., 1980.
3.3.4. Determination of Fungal Frequency %age
The fungal frequency was determined using the following formula for tissue inoculation:
Fungal frequency (%) =Number of colonies of a particular fungus × 100 Total no. of colonies of all fungi in 90 mm petridish
3.3.5. Purification of Fungal Cultures
Cultures of individual fungi were purified using single spore technique. The spores were observed through Olympus stereoscope and were picked with the help of very thin wire inoculating needle and placed in 90 mm petridish containing
PDA. The pure cultures were preserved in mineral oil for further studies.
3.4. RESULTS
3.4.1. Plant Parts Wise Fungal Frequency %age
The plant parts wise % frequency of different fungi associated with declining trees is given below.
3.4.1.1. Mycoflora from citrus decline affected trees
Six fungi (Nattrassia mangiferae, Fusarium sp., Botryodiplodia theobromae, Alternaria sp., and Phytophthora sp.) were isolated from different parts (twigs, branches, bark, stem at collar region and roots) of affected samples of citrus trees from randomly selected villages. In Chak No. 26 NB of tehsil Bhalwal of district SargodhaN. mangiferae was found at maximum frequency (40%) in the bark of tree followed by 22% in stem at collar region, 7% on branches and only 3% frequency was observed from the root samples. Fusarium sp. frequency was maximum (12%) from both branches as well as stem at collar region followed by 97
11% from both twigs and roots. However no Fusarium sp. colony appeared from bark samples. Botryosphaeria sp. was isolated in maximum frequency (39%) twigs,
27% from branches and minimum of 17% from bark. But it was absent in other plant parts. Lasiodiplodia theobromae frequency was maximum from branch samples (34%), from stem at collar region 27 %, from twigs 24%, 12 % from bark and only 2% from roots. Alternaria was mostly found in twigs(14%), 4% from branch samples, 12% from bark and of minimum 6% from the roots. However, the fungus Phytophthora sp. (12%) was only recovered from roots (Fig.3.1). Almost similar trends were observed from other randomly selected villages i.e. Chak 8 NB,
Chak 10 NB, Chak 56 NB, Chak 112 NB and Chak 27 SB as shown in Fig.3.2, 3.3,
3.4, 3.5 and 3.6 respectively.
3.4.1.2. Mycoflora from mango decline affected trees
From mango decline affected trees, samples were taken from six randomly selected villages (Basti Nandla, Chah Nizam Wala, and Basti Ban Bosan of district
Multan and Chak 22 A, Chak 2 P and Mianwali Qureshian of district Rahim Yar
Khan). From the infected trees, six fungi were found associated with different plant parts in varying frequencies. These fungi associted with declining mango trees were N. mangiferae, Fusarium sp., Ceratocystis fimbriata, Lasiodiplodia theobromae, Alternaria alternata and Phytophthora species. The plant wise percentage frequencies of these fungi are given in Fig.3.7, 3.8, 3.9, 3.10, 3.11 and
3.12.
3.4.1.3 Mycoflora from guava decline affectedtrees
Similarly from decline affected guava trees sampling was done in in three randomly selected locations situated in districts of Kasur, Sheikhpura and Nankana 98
Sahib. Six fungi viz. N. mangiferae, Fusarium sp, Botryosphaeria sp, L. theobromae, Alternaria alternata and Phytophthora sp. were isolated.
The percentage frequencies of different fungi isolated from different plant parts of decline affected guava trees from Sehjowal (Tehsil Pattoki of district
Kasur) are given in Fig.3.13. N.mangiferae frequencywas maximum (14%) from bark samples followed by branches (8%) and minimum7% from twig samples.
Fusarium sp. frequency was maximum from roots (13%)followed by 9 %
(branches) and minimum 5% from stem at collar region. The % frequency of
Botryosphaeria sp. was maximum (16%) from branches followed by 6% from twig samples. Lasiodiplodia theobromae frequency was maximum (27%) from stem at collar region followed by branches (15%), twigs (12%) and minimum 3% from bark samples. Likewise, Alternaria sp. frequency maximum (12%) was found from all branches, barks and stem at collar region and minimum 11% from root samples.
The fungus Phytophthora sp. was only recovered from roots (4%).
The percentage frequencies of these six fungi from other two selected locations more or less followed the similar trends and are given in Fig.3.14 and
3.15. The culture and conidia of some of these fungi are given in Fig. 3.16 (a and b), 3.17 (a and b), 3.18 (a and b) and 3.19 (a and b).
Another important point was that in case of citrus and guava there was not any record of Ceratocystis sp. which was solely isolated from mango. It does not mean that it may not be found in citrus or guava but it may be present in some stage of the same genera. The results conclude that the most common fungus found in all types of decline is Nattrassia mangiferae.
99
45
40 Twig
35 branches 30 Bark 25 Stem at collar region 20
15 Roots 10
5
0
Fig.3.1: Plant parts wise fungal frequency %age from decline affected citrus trees at Chak No. 26/NB tehsil Bhalwal, Sargodha.
100
25 Twig 20 branches
15 Bark Stem at collar region 10 Roots 5
0
Fig.3.2: Plant parts wise fungal frequency %age from decline affected citrus trees at Chak No. 8 NB, tehsil Bhalwal of district Sargodha.
101
45 Twig 40 35 branches 30 Bark 25 20 Stem at collar region 15 Roots 10 5 0
Fig.3.3: Plant parts wise fungal frequency %age from decline affected citrus trees at Chak No. 10 N/B, tehsil Bhalwal of district Sargodha.
102
35
30
25
20
15 Twig branches 10 Bark 5 Stem at collar region 0 Roots
Fig.3.4: Plant Parts wise fungal frequency %age from decline affected citrus trees at Chak No. 56/NB of tehsil and district Sargodha.
103
45 40 Twig 35 branches 30 25 Bark 20 Stem at collar region 15 10 Roots 5 0
Fig. 3.5: Plant parts wise fungal frequency %age from decline affected citrus trees at Chak No. 112/NB of tehsil and district Sargodha.
104
35 Twig 30 branches 25 Bark 20 Stem at collar region 15 Roots 10
5
0
Fig.3.6. Plant parts wise fungal frequency %age from decline affected citrus trees at Chak No. 27 SB of tehsil and district, Sargodha.
105
25 Twig
20 branches
15 Bark Stem at collar region 10 Roots 5
0
Fig.3.7: Plant parts wise fungal frequency %age from decline affected mango trees in Basti Nandla, Multan.
106
30 Twig 25 branches 20 Bark 15 Stem at collar region 10 Roots 5 0
Fig.3.8: Plant Parts wise fungal frequency %age from decline affected mango trees in Chah Nizam Wala, Multan.
107
25 Twig 20 branches
15 Bark Stem at collar region 10 Roots 5
0
Fig.3.9: Plant parts wise fungal frequency %age from decline affected mango trees in Basti Band Bosan, Multan.
108
30 Twig 25 branches 20 Bark 15 Stem at collar 10 region Roots 5
0
Fig.3.10:Plant parts wise fungal frequency %age from decline affected mango trees at Chak 22A, tehsil Liaqatpur of district Rahim Yar Khan.
109
30 Twig 25 branches 20 Bark 15 Stem at collar region 10 Roots 5
0
Fig.3.11: Plant parts wise fungal frequency %age from decline affected mango trees at Chak 2P, tehsil Khanpur of district Rahim Yar Khan.
110
35 Twig 30 branches 25 Bark 20 15 Stem at collar region 10 Roots 5 0
Fig.3.12: Plant parts wise fungal frequency %age from decline affected mango trees at Mianwali Qureshian, tehsil and district Rahim Yar Khan.
111
30 Twig 25 branches 20 Bark 15 Stem at collar region 10 Roots 5 0
Fig. 3.13: Plant parts wise fungal frequency %age from decline affected guava trees at Sehjowal, tehsil Pattoki of district Kasur.
112
35 Twig 30 25 branches 20 Bark 15 Stem at collar 10 region 5 0
Fig.3.14: Plant parts wise fungal frequency %age from decline affected guava trees at Faizpur, tehsil Ferozewala of district Sharaqpur.
113
40 Twig 35 branches 30 25 Bark 20 Stem at collar 15 region 10 5 0
Fig.3.15: Plant parts wise fungal frequency %age from decline affected guava trees at Adda Pul Torian of tehsil and district Nankana Sahib.
114
Fig.3.16 (a)
Fig.3.16 (b)
Fig.3.16: (a) and (b) Culture and macrospores of Fusarium sp. 115
Fig.3.17 (a)
Fig.3.17 (b)
Fig.3.17: (a) and (b): Culture and conidia of Ceratocystis sp.
116
Fig.3.18 (a)
Fig.3.18 (b)
Fig.3.18: (a) and (b): Cultural and conidia of Lasiodiplodia theobromae.
117
Fig.3.19 (a)
Fig.3.19 (b)
Fig. 3.19: (a) and (b): Culture and spores of Nattrassia mangiferae.
118
3.5. DISCUSSION
A number of fungi have been isolated from different decline affected trees and different plant parts. These include Botryodiplodia, Botryosphaeria, Nattrassia mangiferae, Fusarium, Alternaria, Pythium, Phytophthora and Ceratocystis species
(Fateh et al., 2006; Kazmi et al., 2007; Fateh et al., 2016, 2017). These fungi have been found infecting the trees singly or in combination (Anwar et al., 2012). It has been observed that Botryodiplodia, Botryosphaeria and Nattrassia mangiferae were mostly found in the twigs and branches of all affected trees. They may be the cause of twig blight and branch die back in the affected trees (Safdar et al., 2015;
Mirzae et al., 2003; Atta and Aref, 2013). Another probable reason for their co- existance is their common lineage (Crous and Palm, 2004). Nattrassia and
Botryodiplodia were also observed on the bark as well as on the inner side of the bark where bark splitting occurred. Therefore, they might have an additional role in bark splitting. Fusarium, Ceratocystis have been found in the vascular tissues of the plants to stop the nutrients inflow and cause the wilting symptoms. However, it is impotant to note that Ceratocystis was only found from the vascular tissues of mango but it was not isolated from citrus and guava (Mirzaee et al., 2003; Miskita et al., 2005; Fateh et al., 2006 and Masood et al., 2010;).
Maximum frequencies of N. mangiferae and B. theobromae recorded in the present study agreed with those described by Giha, (1975); Farr et al. (2005) and
Fateh et al.(2016). It seems that infection by these two fungi has reached an epidemic stage. This is much evident from the host range of these fungi.
Especially, the fungus N. mangiferae caused branch wilt of Banyan trees (Ficus beneghalensis) Khartoum, Sudan (Giha, 1975); top dying and mortality of Gmelina 119
arborea in India (Harsh and Tiwari, 1992). The fungus had an endemic attack on
Eucalyptus camaldulensis and on citrus trees in Arizona (Sigler et al., 1997); caused butt rot of Zanthoxylum bungeanum in China (Cao and Wang, 1989). N. mangiferae invaded number of fruit trees including almond, peach, plum, guava and forest trees like Eucalyptus sp., Populus sp. and Pinus sp. in Iraq (Al Zarrari et al., 1979;Shawkat et al., 1979). The continuing victims of the fungus include lemon trees (gummosis) in Lebanon (Hartmann and Niehaus, 1974) and bananas
(Tip rot) in Jamaica (Meredith, 1963; Jones and Stover, 2000). In USA, the canker of Pacific madrone (Arbutus menziesii) (Davis and DeVay. 1975 and Far et al.,
2005) and foliar disease of strawberry trees (Arbutus unedo) in Europe
(Tsahouridou and Thanassoulopoulos, 2000) was also reported to be caused by the fungus. In Africa it attacked cassava and white yam (Msikita et al., 1997) and the most importantly the fungus has been reported to cause skin and nail diseases and fungal keratitis in humans (Kindo et al., 2010). Being ubiquitous in distribution, it is not sparing trees in Pakistan as well.Although the fungus has not been studied frequently, yet few scientists have reported it on citrus, mango and guava (Fateh et al., 2010; Anwar et al., 2012; Fateh et al., 2016, 2017)
The high frequencies of N. mangiferae in the tropical fruits such as mango and guava are also due to the wide range of temperature these fungi can withstand.
For example Namsi et al.(2010) reported that N. mangiferae grew well within the range of 20 to 40oC. Sehlar et al. (1997) and Mahmood et al. (2002) showed similar results for the growth of B. theobomae.
Another important reason for high frequencies of these fungi is that in the sampling area there were large overlapping trees. This affects the frequencies in 120
two ways: firstly, the large trees with overlapping branches are not the well managed trees i.e. they do not receive proper pruing or protective spray regimes.
Thus the opportunistic fungi found their way through possible wounds due to ploughing or other such cultural practices and invaded the trees without any danger to their own survival (Mirzae et al., 2003). Secondly, the large trees are mostly old age or senescence is itself a contributing factor in tree decline (Khaskheli et al.,
2011; Anwar et al., 2012).
In case of mango, Ceratocystis sp. is unique, as it was not found in both citrus and guava decline affected orchards even though the sampling parts were same for all target fruit trees. One reason can be the resistance of these plants towards this fungus which is yet to be studied or they still did not come in touch with the fungus and are safe. However, across the globe, the fungus Ceratocystis is known to cause mango and many forest tree decline as well as decline in field crops. The fungus for the first time was reported in Pakistan by Fateh et al. (2006) during sampling in mango orchards of Sindh province. Later, the fungus has been repeatedly reported by the relevant scientists in mango as well as shisham (Masood et al., 2010; Safdar et al., 2012; Fatehet al., 2016). Like N. mangiferae and B. theobromae, Ceratocystis genera also penetrate the plants through wounds and establish itself in the xylem vessel, it clogs the xylem and the sap is collected in the small pockets. These pockets later rupture and a thick dark color liquid oozes out which is the characteristic of Ceratocystis sp. (Fateh et al., 2006;Al-Adawi et al.,2006). Unlike other fungi, the fungus Ceratocystis likes low temperatures. Its infection starts in the mid of October to the end of February which are mostly cool 121
months in Pakistan. Hence, its management is possible as October to January is mostly the dormant period of mangoes in the country.
The present study was very significant to report the similar pathogens in citrus, mango and guava decline as this will encourage, similar management pattern for similar fungi in all trees. However, special care will be required in case of mango in future that the deadly fungus Ceratocystis sp. may not enter other fruit orchards from mango ones. This also provides a complete profile that which part of the plant is occupied by which fungi and to devise management accordingly.
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Chapter 4
ROLE OF NATTRASSIA MANGIFERAE AND OTHER DECLINE CAUSING FUNGI IN SYMPTOMS DEVELOPMENT 4.1. INTRODUCTION
There are four major fungi, which have been consistently reported to cause the tree decline in fruit trees, forest and other ornamental plants. These fungi include Nattrassia mangiferae,Ceratocystis sp., Lasiodiplodia theobromae, and
Fusarium sp. The symptoms produced by the inoculation of these fungi alone or in combination are dieback, gummosis, bark splitting, canker formation and mortality of the tree (Kazmi et al., 2007; Fateh et al., 2006).
Different scientists used different methods for the confirmation of pathogenicity of these fungi associated with decline (Khanzada et al., 2004; Urbez-
Torres et al., 2008; Masood et al., 2010). Some of the scientists used green house potted plants and others used branches of the mature trees. In green house potted plants the plugs from fresh cultures were inoculated through flap method, stem injury method and root injury method. In case of mature trees, branches have been artificially inoculated through flap methods (Mirzaee et al., 2003).
In Oman, citrus decline study was conducted by artificial inocultion of acid lime and sweet lime seedlings of age six month to one year. The seedlings were apparently healthy before inoculation. The procedure was done by making injury at the base line of the seedlings and insertion of 5 mm mycelial plug in it that has been taken from 5-6 day-old culture of the fungi grown on potato dextrose agar.
The plugs were wraped by moist cotton to avoid drying of the mycelium or culture.
Seedlings were placed in completely randomized design in a glasshouse under
25oC. The test was replicated 5 times and the symptoms were observed on
97 123
seedlings on weekly basis for 3-4 months for the development of gum, dieback or death. Re-isolations were made to confirm the association Gof the inoculated fungi with the observed symptoms. The isolations were done as explained previously
(Al-Sadi et al., 2010).
Similar studies have also been conducted on mango for C. fimbriata and L. theobromaecausing mango decline. Individual as well as combine inoculation to healthy mango seedling was done in five replications for each treatment under completely randomized design (CRD) in a green house. For inoculation 5mm plug of fungi growing on PDA was placed in slanting cuts under the bark with sterilized scalpel and then covered with parafilm (Mullen et al., 1991). In case of control, only agar slant without any fungal growth was used for insertion. The pathogenicity was confirmed by re isolation of the inoculated fungi (Masood et al.,
2011).
To conduct the pathogenicity for guava decline, detached guava twigs were used for inoculation of the fungi. A moist chamber was developed by placing a blotter sheet on a plastic tray (50 × 40 × 7.5 cm). Twigs of 8‒10 cm length and
2‒3 cm thickness were cut from healthy branches, thouroughly washed twice with distilled water, dried on sterilized blotting paper and re-sterilized with 2% sodium hypochlorite. Twigs were injured with the help of a cork borer. Fungal disks from the respective culture were placed in the holes made as well as uninjured sites of twigs. The uninoculatedtwigs served as control. There were three replications with five twigs per replication. The twigs in trays were incubated in a growth chamber at
28 ± 2°C. The observations were recorded regularly for symptoms or fungal lesion development (Shah et al., 2010). 124
The evaluation of symptoms during pathogenicity has been reported in many different ways. Some of the scientists evaluated by using different rating scales, while others just described the symptoms produced. However, the pathogenicity was confirmed after re-isolation of pathogens from artificially inoculated symptomatic plants.
The aim of present study was to determine the role of Nattrassia sp. in causing decline along with other fungi. Therefore, the pathogenicity of four selected fungi was done individually and in combination which have been associated with decline symptoms directly or indirectly. These fungi included
Nattrassia sp., Fusarium sp., Ceratocystis sp. and Botryodiplodia theobromae.
4.2. REVIEW OF LITERATURE
The fungal involvement in citrus decline was found in Oman. Among these the most common were Lasiodiplodia hormozganensis, L. theobromae and
Fusarium solani. On artificial inoculation of these common fungi, acid lime and sweet lime seedlings reproduced decline and gummosis symptoms. The nursery studies on sweet lime showed association of 12 fungal species, which is an evidence that nurseries act as a main source for some citrus pathogens (Al Sadi et al., 2010).
Abbasher et al. (2013) reported that the fungus Nattrassia mangiferae
(Nattrass) is considered to be the pathogen of branch wilt of citrus, one of the most hazardous diseases that spread in Wad Medani province, Gezira state, Sudan. The survey for the prevalence of fungi isolated from the branch wilt disease samples in
Wad Medani area revealed that that there were 2 different isolates of N. 125
mangiferae. The 1st one was isolated from ficus (Ficus nitida) and the 2nd was isolated from lime (Citrus aurantifolia Swingle.).
A study was conducted to observe the synergistic effect of the fungus
Fusarium semitectum Berk & Rev. and a nematode Tylenchulus semipenetrans
Cobb. was determined singly and in combination on citrus. Maximum reduction in leaves, root length, shoot length, fresh shoot weight, fresh root weight, stem diameter and number of feeder roots were observed in the plants having combined treatment of F. semitectum and T. semipenetrans. Thus, existence of synergistic relationship between F. semitectum and T. semipenetrans in citrus decline was confirmed (Safdar et al., 2013).
Masood et al., 2011 conducted a study to identify the association of pathogenic fungi with mango quick decline tree and the bark beetle. From diseased tree as well as from H. mangiferae, the most frequently isolated fungi were
Lasiodiplodia theobromae, Ceratocystis fimbriata and Phomopsis sp. The fungi were re-isolated from artificially inoculated and symptomatic mango plants. After six months of inoculations, disease symptoms i.e., wilting, oozing and black streaks were developed which showed significant differences among all treatments.
Similar research was done in Oman as well on mango sudden decline.
Affected mango trees showed wilting that began on one side and later spread to involve the entire tree. Trees exude amber-coloured gum from the bark of their trunks or branches and vascular tissues are discoloured. It was found that vascular wilt pathogen Ceratocystis fimbriata caused decline disease in combination with Lasiodiplodia theobromae. Isolates of these fungi from affected trees, caused 126
infection on artificially inoculated seedlings and later recovered from these (Al
Adawi et al., 2006).
Arif (2013) reported the fungi causing mango and guava decline in
Pakistan. These includedCeratocystis fimbriata, Botryodiplodia theobromae,
Fusarium solani, Nattrassia mangiferae and Fusarium oxysporum f.sp psidii. All of these fungi proved to be pathogenic when inoculated on mango and guava plants.
4.3.MATERIALS AND METHODS
4.3.1. Pathogenicity Tests
To test the reponse of various fungi isolated from their respective hosts i.e. citrus, mango and guava artificial inoculation was done individually as well as in combination to potted plants of 1 and half to 2 years old, visually healthy and taken from reliable nurseries. The pathogenicty tests were done under green shade of
NARC, Islamabad. The inoculation was done through flap method and root injury method.
4.3.1.1.Flap method
In the flap method a T shape cut was given in the bark of plants and 2 plugs of 5 mm from freshly growing cultures of test fungi were placed by opening T- shape cut and then wrapped with the help of parafilm. As a control of this method plugs from PDA without cultures were inserted.
4.3.1.2. Root injury method
In this method the plants' roots were injured with the help of sterilized knife and then covered with soil. Spore suspension (5 plugs of 5mm from fresh cultures of fungi were mixed in 200 ml sterilized distilled water) was applied as soil drench. 127
In this method the spore suspension of each fungus was mixed and stirred well before applying as soil drench.
The treatments for citrus in both flap and root injury methods were as follows:
T1= N. mangiferae
T2= B. theobromae
T3= Nattrassia + B. theobromae
T4= 5 mm plug from PDA (Control)
The treatments for mango in both flap and root injury methods were as follows:
T1= Ceratocystis sp.
T2= Nattrassia sp.
T3= Botryodiplodia theobromae
T4= Fusarium sp.
T5= Nattrassia + Ceratocystis
T6= Ceratocystis + B. theobromae
T7= Ceratocystis+ Fusarium
T8= Nattrassia+ B. theobromae
T9= Nattrassia + Fusarium
T10= B. theobromae+ Fusarium
T11= Nattrassia + Ceratocystis + B. Theobromae
T12= Nattrassia + Fusarium + B. Theobromae
T13= Nattrassia + Fusarium + Ceratocystis
T14= B. Theobromae + Fusarium + Ceratocystis 128
T15= Nattrassia + Ceratocystis + B. Theobromae + Fusarium
T16= 5 mm plug from PDA (Control)
The treatments for guava in both flap and root injury methods were as follows:
T1= N. mangiferae
T2= B. theobromae
T3= Fusarium sp.
T4= Nattrassia + B. theobromae
T5= Nattrassia + Fusarium
T6= Fusarium + B. Theobromae
T7= Nattrassia +B. Theobromae + Fusarium
T8= 5 mm plug from PDA (Control)
Each of the above treatment was tested in triplicate and a range of symptoms was observed on citrus, mango and guava seedlings over a period of two months.
4.4.RESULTS
The inoculation results of different fungi by flap method (Fig.4.1) on 1 to 2 years citrus potted plants have been shown in the Table 4.1. The results indicate that when Nattrassia sp. was inoculated alone (T1), bark splitting, stem canker and gummosis appeared but the plants survived. On inoculation of Botryodiplodia theobromae alone (T2), stem canker and gummosis was shown without mortality of the plants. The combination of Nattrassia sp + Botryodiplodia theobromae (T3) showed similar symptoms as N. mangiferae alone. The control plants (T4) without inoculation of any fungi only produced minor gummosis. 129
The inoculation results of different fungi by root injury method (Fig.4.2) through soil drenching the fungal spore suspension on citrus plants have been shown in the Table 4.2.
It was observed when Nattrassia sp. was inoculated alone (T1), bark splitting and gummosis were observed but plants survived. On inoculation of B. theobromae alone (T2), showed only gummosis. The combination of N. mangiferae+ B. theobromae (T3) also showed same results as N. mangiferae alone.
The control plants (T4) without inoculation of any fungi only produced gummosis.
Pathogenicity of different fungi on potted mango plants by flap method is given in Table 4.3. The results indicated that T1, T5, T6, T7, T13, T14 and T15 produced all symptoms of decline i.e. leaf drooping, bark splitting, stem canker, stem gummosis and the final mortality. In all of these cases Ceratocystis sp. was either inoculated alone or in combination with other fungi. In case of other treatments some of the decline symptoms were observed but no mortality was recorded. However, in case of control plants only stem gummosis was seen that may be due to the injury done for inoculation.
Mango plants in case of root injury inoculation method almost showed similar results to that of flap method except, in case of Fusarium sp. inoculated alone also caused mortality of seedling (Table 4.4).
Guava plants response to different fungal inoculations was same as that of mango. Plants of T7 in both flap and root injury method showed all decline symptoms and eventually died. Other treatments similar to mango showed some of the decline symptoms but no mortility occured.
130
Table 4.1: Response of citrus plants against artificial inoculation of N. mangiferae
and B. theobromae using flap method.
Treatments Decline symptoms in citrus(flap method)
Leaf Bark Stem Stem Mortality drooping splitting canker gummosis
T1 - + + + - N. mangiferae (Nm) T2 - - + + - B. theobromae (Bt) T3 - + + + - Nm + Bt T4 - - - + - Control + = Symptoms present ; - = Absent
Table 4.2: Response of citrus plants against artificial inoculation of N. mangiferae
and B. theobromae using root injury method.
Treatments Decline symptoms in citrus(Root injury)
Leaf Bark Stem Stem Mortality drooping splitting canker gummosis T1 - + - + - N. mangiferae (Nm) T2 - - - + - B. theobromae (Bt) T3 - + - + - Nm + Bt T4 + - - + - Control + = Symptoms present ; - = Absent
131
Table 4.3: Response of mango plants against artificial inoculation of Caratocystis
sp., N. mangiferae, B. theobromae and Fusarium sp. using flap
method.
Treatments Decline symptoms in mango(flap method)
Leaf Bark Stem Stem Mortality drooping splitting canker gummosis T1 + + + + + Ceratocystis sp.(Cs) T2 - + - + - N. mangiferae (Nm) T3 - + + + - B. theobromae (Bt) T4 + - + + - Fusarium sp. (Fu) T5 + + + + + Nm + Cs T6 + + + + + Cs + Bt T7 + + + + + Cs + Fu T8 - + + + - Nm + Bt T9 - + + + - Nm + Fu T10 - - + + + Bt + Fu T11 - - - + - Nm + Cs + Bt T12 + + + + - Nm + Fu + Bt T13 + + + + + Nm + Fu + Cs T14 + + + + + Bt + Fu + Cs T15 + + + + + Nm + Bt + Cs + Fu T16 - - - + - Control + = Symptoms present ; - = Absent
132
Table 4.4: Response of mango plants against artificial inoculation of Caratotocystis
sp., N. mangiferae, B. theobromae and Fusarium sp. using root injury
method.
Treatments Decline symptoms in mango(root injury)
Leaf Bark Stem Stem Mortality drooping splitting canker gummosis T1 + + + + + Ceratocystis sp.(Cs) T2 - - - + - N. mangiferae (Nm) T3 - + - + - B. theobromae (Bt) T4 + + + + + Fusarium sp. (Fu) T5 + + + + + Nm + Cs T6 + + + + + Cs + Bt T7 + + + + + Cs + Fu T8 - + + + - Nm + Bt T9 + + + + - Nm + Fu T10 + + + - + Bt + Fu T11 + + + + + Nm + Cs + Bt T12 - + + + - Nm + Fu + Bt T13 + + + + + Nm + Fu + Cs T14 + + + + + Bt + Fu + Cs T15 + + + + + Nm + Bt + Cs + Fu T16 - - - + - Control + = Symptoms present ; - = Absent
133
Table 4.5: Pathogenicity of fungi associated with guava decline by using flap
method inoculation.
Treatments Decline symptoms in guava (Flap method)
Leaf Bark Stem Stem Mortality drooping splitting canker gummosis T1 - + - + - N. mangiferae (Nm) T2 - + + + - B. theobromae (Bt) T3 + - + + - Fusarium sp. (Fu) T4 - + + + - Nm + Bt T5 + + - - - Nm + Fu T6 + + + + - Fu+ Bt T7 + + + + + Nm + Bt + Fu T8 - - - - - Control + = Symptoms present ; - = Absent
134
Table 4.6:Pathogenicity of fungi associated with guava decline by using root injury
method of inoculation.
Treatments Decline symptoms in guava(root injury)
Leaf Bark Stem Stem Mortality drooping splitting canker gummosis T1 - + - + - N. mangiferae (Nm) T2 - + - + - B. theobromae (Bt) T3 + - - + - Fusarium sp. (Fu) T4 - + + + - Nm + Bt T5 + + - + - Nm + Fu T6 + + + + - Fu+ Bt T7 + + + + + Nm + Bt + Fu T8 + - - + - Control + = Symptoms present ; - = Absent
135
Fig.4.1: Flap method of inoculation.
Fig.4.2: Root injury method of inoculation. 136
The response of plants in all cases showed that Nattrassia alone cannot cause the mortality of plants. However, in combination with other fungi it can produce all the symptoms we observe in the decline affected trees of citrus mango and guava. Moreover, the fungus Ceratocystis sp. has the ability to cause the mortality of plants which is more common in case of mango.
4.5. DISCUSSION
The present study was done to evaluate the role of N. mangiferae incausing decline among other popular fungi that has been repeatedly reported from various declining trees. It was necessary to include those fungi in pathogenicity experiment as various scientists reported the cause of decline differently, for example
Mahmood et al.(2002) reported that the cause of decline is B. theobromae. Similar results were shown by Khanzada et al.(2005). However, Fateh et al. (2006); Al
Adawi et al. (2006) and Masood et al. (2010) reported the cause of the disease was
Ceratocystis sp. Similar results were also reported for citrus and guava decline, where the role of B. theobromae and Fusarium sp. were given more importance than other fungi (Safdar et al., 2015; Bokhari et al., 2008). There was another school of thought who has reported that in case of guava decline, there is an association of Fusarium sp. and Meloidogyne sp. (Gomes et al., 2012).
The similar behavior of N. magiferae and B. theobromae upon individual inoculation for producing symptoms irrespective of the hosts once again confirms that they inherit a common lineage as discussed in Chapter 3. It is important to discuss here that most of these fungi are endophytic. Endophytic fungi live symbiotically with the majority of plants by entering their cells (Hirsch and Braun, 137
1992) and are utilized as an indirect defence against herbivores. This means that probably when the fungus is alone within a plant, it plays its symbiotic part, but when combined with other fungi it becomes aggressive and damages plants. In the present study, individual fungi except Ceratocystis were not able to produce mortality at all. However, the combinations of these fungi caused mortality in the green house potted plants; these results are in agreement with those of Fateh et al.(2006); Al Adawi et al.,
(2006); Masoodet al. (2010) and Fateh et al. (2016).
Among the two methods used for pathogenicity, root injury was the most effective for infection development and causing the mortality of plants. This also confirms the findings that the decline causing fungi and in particular the
Ceratocystis sp. when enter the plants through wounds during cultural practices such as plowing, hoeing and weeding, invade roots, move to xylem part where they block the intake of water and nutrient inflow. Thus, cause permanent wilting or mortality of the tree (Fateh et al., 2006; Al Adawi et al., 2006; Kazmi et al, 2007;
Masood et al., 2010 and Fateh et al., 2016). This also suggests that when some screening studies are planned especially for varietal resistance against Ceratocystis sp. or other decline causing fungi, the root injury method should be adopted.
The fungus Fusarium sp. in case of mango has been able to cause mortality but in only one case when it was inoculated through root injury method. This once again strengthens the statement that the root injury method of inoculation was the most effective to cause disease which enabled Fusarium as well to cause mortality.
In case of guava the death caused by Fusarium was already reported by Pandey and
Dwivedi (1985). 138
The fungus N. mangiferae in field conditions enters the plants through wounds not only from the branches through bark cracks but also through stem and injured roots. This fungus in the presence of other fungi such as Fusarium sp., B. theobromae, Ceratocystis sp. plays an equal role for creating a situation where the plants normal functioning is disturbed in target fruits i.e. mango, citrus and guava as reported by Safdar et al.(2015) and Fateh et al. (2016).
139
Chapter 5
SOME PHYSIOLOGICAL STUDIES OF FUNGI ASSOCIATED WITH DECLINE 5.1. INTRODUCTION
Basic knowledge about the pathogen, its survival, spread and other related aspects are very important for the better management of any disease. Physiological studies include the study of factors that contribute towards the physiological development of the fungi. These factors can be different sources of carbon and nitrogen by using different culture media to observe the best growth on a particular medium (Walker and White, 2005).
It is a known fact that physiological development offungi is largely affected by environmental factors such as temperature, light and pH. Most of the time these studies are the part of epidemiology and are conducted in vitro (Charlie and
Watkinson, 1995).
The factors that influence physiological development of fungi include the optimum ranges of temperature, light, pH and carbon sources that can either favour or inhibit the growth of fungi. Therefore, these studies lead to develop the management strategies for different diseases (Hubbali et al., 2010).
As the effects of these physiological factors have not been studied on decline inciting fungi in Pakistan. Therefore, the present studies were conducted to assess the effects of different physiological factors on the growth of the fungi isolated from citrus, mango and guava with special emphasis on N. mangiferae.
5.2. REVIEW OF LITERATURE
Physiological studies have been done on decline causing fungi on several
114 140
different hosts. For example Lasiodiplodia theobromae, causing decline of common tea (Camellia sinensis) does not sporulate or poorly sporulates on potato dextrose agar (PDA). Therefore, a study was planned to determine the effects of culture media, carbon source, nitrogen source, temperature, pH and light on mycelial growth and sporulation. Among several carbon sources tested, glucose and sucrose were found superior for growth. The addition of tea root extract to potato dextrose agar medium was found to be the most suitable for mycelial growth and sporulation of L. theobromae. It was found that the fungus grew at temperatures ranging from 20 to 36 oC, with optimum growth at 28 oC and no growth was noted at 40 oC. There was no significant effect of different light periods on growth of L. theobromae, however, light enhanced sporulation. The fungus grew at pH 3.0-8.0 and optimum growth was observed at pH 6.0. Tea root extract supplemented potato dextrose agar medium with pH 6.0 was the most suitable for production of conidia of L. theobromae at 28 oC (Saha et al., 2008).
However, Kausar et al. (2009) reported that Optimum temperature for both
Fusarium solani and Lasiodiplodia theobromae fungi was 25 ºC. They found that continuous light supported growth more as compared to darkness (for 24 hours) and 12 hours light plus 12 hours darkness for the growth of fungi. Among two media tested for both fungi, grew maximum on Poatao Dextrose Agar (PDA) medium but least grew on the water agar (WA) medium.
According to Alam et al. (2001) Botryodiplodia theobromae grew and sporulated well at 15-40°C, but optimum between 25-30oC. Among culture media
PDA showed maximum mycelial growth and sporulation. However, no growth was observed at 10 and 45°C. 141
Similarly,Ceratocystis fimbriata is a decline cauing pomegranate decline, grew well in all most all pH ranging from 2.0 to 11.0. It indicates that the fungus can survive in all soil types and locations. Maximum growth has been recorded at a pH of 7.5 with no significant difference with that on pH 7 and 8. The fungal growth was inhibited below pH 5.5 and above pH 9.0. Lowest growth was observed at pH
2.0. The maximum growth of the fungus was observed at a temperature of 30oC and failed to grow at 45oC (Sonyal el al., 2015). However, the best growth from other hosts has been obtained at 25oC. This shows that the fungus when isolated from different hosts can behave differently.
Fusarium oxysporum f. sp. psidii and F. solani, cause wilt in guava and are highly variable pathogens. Physiological studies of the fungi revealed that maximum mycelial growth was obtained on PDA followed by ME broth as liquid medium. Maximum sporulation was recorded in oatmeal agar (OMA). The optimum temperature and pH for growth of Fusarium sp. was 28oC and 5.5 respectively. The isolates had differences in their colony growth, mycelial mass, macro-conidia, and micro-conidia produced. These variations were characters of each of the isolates with respect to cultural and physiological characters (Gupta et al., 2010).
Dring a field study on cashew diseases three fungal generaLasiodiplodia,
Fusarium and Pestalotiafound were cultured on Potato Dextrose Agar medium.
The optimum temperature for the growth of Fusarium was 250C while all the temperature treatments 20oC, 25oC and 30oC were found suitable for the growth of
Lasiodiplodia and Pestalotia. Moreover, alternating 12 hours light and12 hours darkness was more suitable for the growth of Fusarium than continuous darkness. 142
The growth of both Lasiodiplodia and Pestalotia were not affected by the light condition.
5.3. MATERIALS AND METHODS
5.3.1.Effect of Temperature, Light, pH and Culture Media on Fungal Growth
The effect of culture media, temperature, light and pH was observed on mycelial growth of fungi including Nattrassia mangiferae, Ceratocystis sp.,
Botryodiplodia theobromae and Fusarium sp. in 90 mm petridish. The study was done to have the basic information of suitable temperature, growth medium, pH and sensitivity to alternate light and dark periods. For this purpose the temperature ranges used were, 20oC, 22oC, 25 oC and 28 oC, the pH ranges 5.5, 6.0 and 6.5.
Light and dark periods of 12 hrs respectively and complete 24 hours of continuous light and continuous dark. The culture media tested were MEA, PDA and SDGAM.
In all cases other than target variable tested all conditions were kept constant. The radial growth was measured by the ruler from the central point of inoculation at the back of 90 mm Petri dish containing culture. Four observations were taken from around and the average was calculated.
5.3.1.1. Effect of culture media.
Four culture media i.e. Potato Dextrose Agar (PDA), Malt Extract Agar
(MEA), Stem Decoction Glucose Agar (SDGA) and Carrot Juice Agar (CJA) were evaluated to find out the most suitable one for the growth of the fungi. Each culture medium was prepared in 1 L of water and autoclaved at 121 oC at 15 psi for 20 min. These were cooled to 45 oC and then poured in 90 mm Petri dishes for solidification. A plug of 5 mm was taken from freshly growing culture of each of the fungi was taken and inoculated to plates containing above mentioned media. 143
The plates were observed for mycelial growth everyday and on 7th day the final reading was taken. The experiment was done in triplicate.
5.3.1.2. Effect of temperature
As mentioned above 5 mm culture plugs were taken by sterilized cork borer from advancing margin of freshly growing target fungal cultures and inoculated on
PDA containing 90 mm petridishes. The incubation was done on PDA at 20oC, 22 oC, 25oC and 28oC for 7 days till the final reading was taken for radial mycelial growth measurement. The experiment was done in triplicate.
5.3.1.3. Effect of light
For the effect of light and darkness on mycelial growth of isolated fungi, 5 mm culture discs were cut with sterilized cork borer from advancing margins of the colonies of isolated fungi and inoculated on PDA plates separately for seven days.
To create condition of darkness carbon paper was used to wrap the petridishes.
Another set of petridishes of fungal culture was without wrap. All petridishes were incubated at 25+1oC in triplicates and alternating light and darkness (12 hours light plus 12 hours darkness) and complete darkness (24 hours) was provided to record the effects in terms of mycelial growth. The mycelial growth in mm was recorded on 7th day of inoculation.
5.3.1.4. Effect of pH
Target fungi were inoculated as bove in petridishes containing PDA and incubated at optimum temperature 25+ 1 oC. The pH of PDA medium was adjusted
5.5, 6.0 and 6.5 with 0.1 N NaOH/HCl with pH meter. Optimum pH was determined by calculating colony diameter on daily bases till final reading on 7th day. 144
5.4.RESULTS
5.4.1. Effect of Culture Media
The culture media had significant effects on the growth of decline causing fungi. Among isolates from citrus, the fungusN. mangiferaeshowed maximum radial growth on MEA followed by PDA which was not significantly different from that of MEA. Minimum growth was recorded on SDGA and CJA. Similarly, there was significant difference in growth of Fusarium sp. on PDA and MEA. The fungi Botryosphaeria sp. and B. theobromae both showed no significant difference in growth on PDA and MEA. The growth on SDGA and CJA was significantly less as compared to PDA and MEA (Table 5.1).
The fungi isolated from declining mango trees had significant differences in growth on different media.N. mangiferae gave maximum growth on MEA medium
PDA. The growth of the fungus was significantly less on SDGA and CJA media
(Table 5.2).
Unlike N. mangiferae isolated from citrus, which showed no significant difference in growth on MEA and PDA media, the isolate from mango showed significant difference in radial mycelial growth on the media. The growth on MEA was significantly higher than on PDA medium. This shows that isolates of same fungus from different hosts can behave differently when growth is tested on same culture media. This is because that fungus can have varying virulence levels on different hosts (Makun et al., 2010)
The isolates of fungi from guava showed that N. mangiferae has significant difference in growth on MEA from rest of the culture media giving the maximum 145
(75 mm) radial growth. Similarly, B. theobromae from guava showed significant difference in growth on PDA and MEA (Table 5.3).
The results in table 5.3 showed that N. mangiferae and Fusarium sp. gave best growth on PDA and MEA. The fungus Ceratocystis sp. grew well on SDGA and CJA. The Botrysphaeria sp. and B. theobromae showed variable responses in growth on different culture media. This also showed that the fungi isolated from mango and guava had resemblance regarding radial mycelial growth on tested culture media. However, the fungal isolates from citrus species behaved differently.
5.4.2. Effect of Temperature on Radial Mycelial Growth
The decline causing fungi behaved differently when grown on different temperatures.
The statistical analysis showed highly significant results regarding the effect of temperature on radial growth. The growth of Nattrassia mangiferae increased from 20oC to 25oC and then declined. Maximum radial growth of all the fungi was recorded at 25oC, below and above this temperature their growth declined (Table 5.4).
All the fungi isolated from mango grew to the maximum at a temperature of
25oC except Ceratocystis sp. which showed maximum growth at 22oC as shown in
Table 5.5.
Similarly, the fungi isolated from guava followed the same growth patterns at different temperature regimes as shown by the isolates from citrus and mango
(Table 5.6).
146
Table 5.1: Effect of culture media on radial mycelial growth of the fungi isolated
from Citrus.
Fungi Culture Media (Average growth in mm)
PDA MEA SDGA CJA
Nattrassia mangiferae 80 e 81 e 74 d 67 c
Botryosphaeria sp. 85 e 84 e 71 d 70 cd
Botryodiplodia theobromae 80 e 79 e 75 d 70 cd
Fusarium sp. 65 c 61 a 57 b 58 b
Data are mean of threereplicates
Table 5.2: Effect of culture media on radial mycelial growth of the fungi isolated
from mango.
Fungi Culture Media (Average growth in mm)
PDA MEA SDGA CJA
Nattrassia mangiferae 69 ef 75 fg 68 e 65e
Ceratocystis sp. 43 a 51 b 55 c 54 c
Botryodiplodia theobromae 84 ef 72 f 71 f 67 e
Fusarium sp. 70 f 65 e 58 cd 58 cd
Data are mean of threereplicates
147
5.4.3. Effect of Light on Radial Mycelial Growth
Light significantly affected the growth of fungi isolated from different hosts. It was found that continuous light or continuous dark did not have any significant effect on the growth of fungi isolated from citrus. However, alternate light and dark periods were found significant for the growth of fungi as shown in
Table 5.7. Similar findings were reported by Alam et al. (2001) and Sharma et al.
(2005).
The fungi isolated from declining mango trees followed the same pattern as shown in Table 5.8. B. theobromae and Fusarium sp. had no significant differences in any case. The results are in line with Rehman et al. (2011).
In case of the fungal isolates from guava the growth of B. theobromae when kept in continuous dark was the minimum. However, it showed maximum growth in alternate light and dark (80 mm) followed by continuous light conditions (75 mm). Thus, it did not show the significant difference in growth when placed in either complete dark or under alternate light and dark hours. From the results it seemed that the fungus had photophobic behavior. The rest of the fungi has no clear significant difference in all cases (Table 5.9).
5.4.4. Effect of pH on Radial Mycelial Growth
The pH had significant effect on the growth of Botryosphaeria sp. it showedmaximum growth at pH 5.5. Similarly,Fusarium sp. had significant difference in growth at pH 5.5 from other pH ranges. The rest of the fungi have no clear significant difference (Table 5.10). 148
N. mangiferae isolated from mango showed significant difference in growth at different pH regimes. At pH 5.5, it gave maximum growth followed by that at pH 6.0 and 6.5 respectively.
Table 5.3: Effect of culture media on radial mycelial growth of the fungi isolated
from guava.
Fungi Culture Media
(Average growth in mm)
PDA MEA SDGA CJA
Nattrassia mangiferae 70 def 75 fg 67 de 65 d
Botryosphaeria sp. 75 fg 78 g 54 ab 51 a
Botryodiplodia theobromae 82h 73 f 70 def 68 de
Fusarium sp. 71 def 64 d 57 bc 58 bc
Data are mean of three replicates
Table 5.4: Effect of temperature on radial mycelial growth of the fungi isolated
from citrus on PDA medium.
Fungi Temperature (oC) (Average growth in mm)
20 22 25 28
Nattrassia mangiferae 80 def 81 def 84 ef 78 cdef
Botryosphaeria sp. 74 cd 78 def 85 f 80 def
Botryodiplodia theobromae 76 cde 80 def 85 f 81 def
Fusarium sp. 43 a 54 b 71 c 68 c
Data are mean of 3 replicates
149
Table 5.5: Effect of temperature on radial mycelial growth of the fungi isolated
from mango on PDA medium.
Fungi Temperature (oC) (Average growth in mm)
20 22 25 28
Nattrassia mangiferae 68c 75d 83e 80e
Ceratocytis sp. 55b 57b 55b 41a
Botryodiplodia theobromae 67c 72cd 84e 82e
Fusarium sp. 41a 55b 74d 69c
Data are mean of three replicates
Table 5.6: Effect of temperature on radial mycelial growth of the fungi isolated
from guava on PDA medium.
Fungi Temperature (oC) (Average growth in mm)
20 22 25 28
Nattrassia mangiferae 64 b 68 bc 81f 75 def
Botryosphaeria sp. 67 bc 70 bcd 79 ef 78 ef
Botryodiplodia theobromae 70 bcd 73 cdf 80 f 75 ef
Fusarium sp. 48 a 53 a 79 ef 65 b
Data are mean of three replicates
150
Table 5.7: Effect of light on radial mycelial growth of the fungi isolated from citrus
on PDA medium
Fungi Light Conditions (Average growth in mm)
12 hrs light+ Continuous Continuous 12 hrs dark light dark
Nattrassia mangiferae 80 d 65 ab 68 ab
Botryosphaeria sp. 77 d 69 bc 75 cd
Botryodiplodia theobromae 80 d 75 cd 70 bc
Fusarium sp. 62 a 68 a 65 ab
Data are mean of threereplicates
Table 5.8:Effect of light on radial mycelial growth of the fungi isolated from
mango on PDA medium.
Fungi Light Conditions (Average growth in mm)
12 hrs light+ Continuous Continuous 12 hrs dark light dark
Nattrassia mangiferae 76 ef 74 ef 71 e
Ceratocystis sp. 56 cd 48 ab 43 a
Botryodiplodia theobromae 79 f 74 ef 74 ef
Fusarium sp. 59d 55 cd 52 bc
Data are mean of threereplicates
151
While, Ceratocystis sp. and Fusarium sp. showed significant difference in growth at pH 6.5.Ceratocystis sp. gave maximum growth at pH 6.5 and Fusarium sp. gave minimum growth at pH 6.5. The fungus B. theobromae followed same pattern as shown in Table 5.11. The results coincide with those of Lathal et al.(2012) and Rehman et al. (2011).
The isolates of N. mangiferae, Botryosphaeria sp. and Fusarium sp. from guava had significant difference in growth at 5.5 pH. All fungi showed maximum growth at pH 5.5 (69 mm, 65 mm and 62 mm respectively).
The fungus B. theobromae had no clear significant difference in growth in any case and grew maximum at pH 5.5 (75 mm) followed by 72 mm and 68 mm respectively at pH 6.0 and 6.5 (Table 5.12).
Results showed that the alternate light and dark conditions have sigfunginificant effect on growth of N. mangiferae, Botryosphaeria sp., B. theobromae and Fusarium sp. However, the fungus Ceratocystis sp. has shown significant growth in contiuous dark condition.
The results regarding pH have shown that all of the fungi like low pH level.
As pH increases the mycelial growth is reduced. The results are somehow similar in citrus and guava. However, in case of mango, Ceratocystis sp. has ascending mycelial growth trend. The lowest pH range 5.5 and 6.0 has significantly reduced growth as compared to pH level 6.5.
Similarities were also found in growth pattern of fungi associated with
Botryosphaeriacae family. This was very astonishing that within the same mango trees the isolated mycoflora have different choices of environmental conditions, especially the fungi belong to Botryospaeriace and Ceratocystis sp. 152
Table 5.9: Effect of light on radial mycelial growth of the fungi isolated from
guava on PDA medium.
Fungi Light Conditions (Average growth in mm)
12 hrs light+ Continuous Continuous 12 hrs dark light dark Nattrassia mangiferae 69 abcd 65 abc 63 ab
Botryosphaeria sp. 70 bcd 64 abc 61 a
Botryodiplodia theobromae 80 e 75 de 72 cd
Fusarium sp. 64 abc 61 a 65 abc
Data are mean ofthree replicates
Table 5.10: Effect of pH on radial mycelial growth of the fungi isolated from citrus
on PDA medium.
Fungi pH of medium (Average growth in mm)
5.5 6.0 6.5
Nattrassia mangiferae 71 ef 68 def 63 cd
Botryosphaeria sp. 74 f 62 cd 59 bc
Botryodiplodia theobromae 70 ef 66 de 65 cde
Fusarium sp. 59 bc 54 b 47 a
Data are mean of threereplicates
153
Table 5.11: Effect of pH on radial mycelial growth of the fungi isolated from
mango on PDA medium.
Fungi pH of medium (Average growth in mm)
5.5 6.0 6.5
Nattrassia mangiferae 79 gh 71ef 68 ef
Ceratocytis sp. 49 a 51a 57 bc
Botryodiplodia theobromae 81h 78gh 74fg
Fusarium sp. 65de 59cd 51ab
Data are mean ofthree replicates
Table 5.12: Effect of pH on radial mycelial growth of the fungi isolated from guava
on PDA medium.
Fungi pH of medium (Average growth in mm)
5.5 6.0 6.5
Nattrassia mangiferae 69 bcd 51a 49 a
Botryosphaeria sp. 65 bc 52 a 51a
Botryodiplodia theobromae 75 d 72 cd 68 bcd
Fusarium sp. 62 b 54 a 51a
Data are mean of 3 replicates
154
5.5. DISCUSSION
It is clear from the results that when the growth of fungal isolates from citrus was tested against various media i.e. PDA, MEA, SDGA and CJA, three fungi among 4 have shown no significant difference in the growth. These fungi are
Nattrassia mangifera, Botryodiplodia theobromae and Botryosphaeria sp. As discussed in chapter 3 that these fungi share a common lineage and hence they showed no significant difference on each of the culture media used for their growth. However, Fusarium sp. growth was significantly different from the rest of the fungi in the study. The significantly shown growth may be due to its slow growing nature already reported in the past (Schroeder et al. 2006; Kang et al.,
2014). The other reason can be its growth response to a selective medium rather than a range of media (Vanwyk et al., 2006; Kang et al., 2014).
It was astonishing to know that N. mangiferae isolates from mango showed completely different behaviour in term of growth within stipulated time i.e. significantly less growth was observed in all of the tested culture media as compared to its isolates from citrus. It is due to the fact that the growth of N. mangiferae in mango is hampered by plant defence mechanisms (Elliott and
Edmonds, 2008) and it is reasonably slow growing in nature. However, further research is needed in this regard. Among other fungi, Ceratocystis sp., showed significantly slow growth on all of the media tested. The difference was also observed in its growth on potato dextrose agar (PDA) and malt extract agar (MEA) but no significant difference was found onstem decoction glucose agar (SDGA) and carrot juice agar (CJA). Ceratocystis sp. is known for its complexity in growth 155
and very choosy in physiological conditions especially temperature (Fateh et al.,
2010; Rehman et al., 2011; Masood et al., 2011). Secondly, culture media like
SDGA and CJA media were particularly developed for the fungus Ceratocystis sp. based on the experience of scientists associated with this fungus (Moller and De
Vay, 1968; Barnes et al., 2003). During the study it was observed that on inoculation of infected tree samples on SDGA and CJA, only the fungus
Ceratocystis sp. was isolated but its growth was observed in bits and patches spread throughout the 90 mm petridish but once re-culturing was done the normal growth was observed in the petri plates and this is the new finding of the study.
There was not much difference in growth among the fungal isolates from guava when compared with mango. This may be due to the fact that guava in Pakistan is grown with similar climate as mangoes. Sometimes, guava is intercropped with mango in orchards (Khushk et al., 2009).
Unlike N. mangiferae isolated from citrus which showed no significant difference in growth on MEA and PDA media, the isolate from mango showed significant difference in radial mycelial growth on the media. The growth on MEA was significantly higher than that on PDA medium. This shows that isolates of same fungus from different hosts can behave differently when growth is tested on same culture media. This is because that fungus can have varying virulence levels on different hosts (Makun et al., 2010).
The response of growth of N. mangiferae isolates from citrus was much interesting. It showed the aggressive growth from 20oC to 25oC and started declining after 28oC. The results are in agreement with those of Jamali and
Banihashemi(2010) and Rehman et al.(2011). It followed similar pattern in growth 156
when isolated from mango as well as guava. However, relatively less growth among the isolates from mango and guava was observed as compared to citrus isolates. The reason can again be the similar climatic areas occupied by mango and guava. It is important to note that the fungus Ceratocystis sp. again preferred a low temperature range as compared to other fungi, in case of mango (Fateh et al., 2006 and Rehman et al., 2011). The results also suggest that fluctuation in temperature regimes in field condition affect both the growth of trees as well as the growth of
Ceratocystis sp. This fact has been supported by Reynolds(1992).
Mycelial growth was seen more in the isolates of Nattrassia when provided alternate light and dark periods as compared to continuous light or continuous dark periods. However, again the isolates from mango and guava did not show significant differences in any condition of light and dark (Elliott and Edmonds,
2003). Nattrassia isolates from citrus were the least affected by the pH range of 5.5 to 6.5. However, in mango and guava the isolates performed well at pH up to 6.00.
The results are contradictory to the actual climatic conditions especially in the mango orchards where soil pH is more than 7 andN. mangiferae keeps growing but the results are in agreement with the findings of Namsi et al. (2010) andEl Atta and
Aref (2013).
157
Chapter 6
MORPHOLOGICAL VARIABILITY AMONG ISOLATES OF COMMONLY FOUND FUNGUS (NATTRASSIA MANGIFERAE)FROM DECLINE AFFECTED CITRUS, MANGO AND GUAVA TREES 6.1. INTRODUCTION
The most common fungi found from decline affected trees are Nattrassia mangiferae and Botryodiplodiatheobromae. These fungi are found in different parts of the plants causing different symptoms. However, the same specie might have some different characteristics when invading different hosts. Mostly these fungi invade twigs and branches of the trees (Al- Adawi, 2002, Asif et al., 2011).
The fungus N. mangiferae belongs to class Coelomycetes. The fungus is polymorphic and has two spore stages, the pycnidial and the arthroconidial stage.
Some anamorphs of Botryosphaeria are similar to Nattrassia both in their behavior on the host plant and morphology. They attack stressed plants especially due to drought or physical wounding. The fungus can cause canker, branch die back, twig die back and blossom blight. Mostly these fungi are endophytic and become aggressive to cause symptoms when the host is under stress conditions (Denman et al., 2003).
N. mangiferae has been reported to cause a number of diseases in different host plants including: branch wilt of walnut, citrus, figs,branch canker of eucalyptus, madrone, acasia and mango, dieback and trunk canker of guava (El-
Atta and Aref, 2013).
The fungus N. mangiferae is basically a wound invading pathogen of forest and fruit trees and has been reported to be the cause of foot canker of Hevea
132 158
brasiliensis seedlings in Sri Lanka. The cultural characteristics and the morphology of the reproductive structures such as thallospores, pycnidia, pycnospores of Hevea isolate from Sri Lanka resembles with the isolate of N. mangiferae obtained from other hosts in different parts of the world (Jayasinghe et al., 1997).
In the year 2000, in the Jiroft region, South-Eastern Iran, N. mangiferae was isolated from both Ficus religiosa, which showed branch die-back and elongated cankers in trunks, and Psidium guajava (guava) trees, which showed branch die- back. The pathogenicity of the fungus was demonstrated by fulfilling Koch's postulates. These diseases are a potential threat to the citrus industry and P. guajava production in the region. This is the first report of N. mangiferae inciting die-back and trunk cankers on F. religiosaand die-back on P. guajava (Mirazeeet al., 2003).
A survey of branch wilt disease was conducted in 2008, 2009 and 2010 in
South Kordofan State. The survey was done on different hosts inclunding citrus, mango and other fruit trees. The tree showed symptoms of dieback, bark cracking and sloughing off, gum exudates on pockets at the base of infected branches.
N. mangiferae was dominant pathogen in the samples collected from branch wilt affected trees. Other opportunistic fungi found were such as Penicillium sp.,
Aspergillus niger and Fusarium oxysporum.
Since N. mangferae has been reported from a variety of hosts causing variety of symptoms, the present study was aimed to see the morphological variations among different isolates of these fungi based on their location, tree and plant parts from which they were isolated with a hypothesis that there may be some morphological variations among the isolates. 159
6.2.REVIEW OF LITERATURE
In Pakistan the fungus C. fimbriata for the first time was reported in Sindh from declining mango trees. It's identification was confirmed by morphological characteristics of perithecia (brown to black with globose base, necks almost 800-
900 µm long with ostiolar hyphae), ascospores (elliptical 4-8 × 2-5 µm, hat shaped) conidiophores (hyaline, septate up to 150 µm long and conidia that were cylindrical, sometimes in chains and truncated at the ends (Fateh et al., 2006).
Lasiodiplodia theobromaewhich is the anamorph of Botryosphaeria rhodina is an opportunistic fungus attacking on more than 500 tree species in the tropics and subtropics. During surveys of different plant diseases in Australia and
Venezuela various isolates were some what different from L. theobromae based upon morphology and subsequently characterized and ITS and EF1-α nucleotide sequences. These were described as three new speciesi.e. L. venezuelensis sp. nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. Theywere easily distinguished from each other (Burgess et al., 2006).
Nattrassia mangiferaeanother opportunistic wound invading fungus of many forest and fruit trees was reported to cause of Hevea brasiliensis foot canker in Sri Lanka. The morphology of the reproductive structure: thallospores, pycnidia, pycnospores of Hevea isolate from Sri Lanka resembled with the isolate of N. mangiferae obtained from other hosts in different parts of the world (Jayasingheet al., 1997).
Elliott and Edmonds in 2003 identified N. mangiferae, causing a canker disease of Pacific madrone, using morphological and molecular methods. Only asexual spores were observed, but sequencing of the ITS region of the ribosomal 160
rDNA places the sexual state in the genus Botryosphaeria. Resembles of the fungus was found with Fusicoccumwhich is anamorphs of closely related Botryosphaeria species and had a similar pathology.
Several pecies of Botryosphaeria affect mango trees and fruit. These are mainly identified on the morphology of the anamorphs, include,Dothiorella dominicana, D. mangiferae (Natrassia mangiferae), D. aromatica and an unidentified species, Dothiorella „long‟. The genus name Dothiorella, however, is acknowledged as a synonym of Diplodia. To achieve isolates representing all four
Dothiorella spp. the anamorphs of known Botryosphaeria sp., were based on conidial morphology and DNA sequence data. The morphological and molecular results confirmed that the fungi previously identified from mango as species of
Dothiorella belong to Fusicoccum. This study provided basis for the identification of Botryosphaeria species from mango, which is important for disease control and to uphold quarantine regulations (Slippers et al., 2005).
In, Iran, a study was conducted using morphological and molecular methods to identify the causal agent of black locust treesdecline. The isolates of the fungus produced arthroconidia, and the fungus identified wasNeofusicoccum mangiferae.Pathogenicity test under the bark produced similar symptoms as in natural infection. Species classification was confirmed by analysis of the internal transcribed spacer (ITS) sequencing. The funus was mostly similar to N. mangiferae(Nazerianet al., 2015).
A fungus resembling the asexual morphs in the family Botryosphaeriaceae was isolated from a fallen leaf of an orchid collected in Thailand. After morphological and phylogenetic analyses it was placed as Neoscytalidium sp. 161
(Huang et al., 2016).
6.3.MATERIALS AND METHODS
6.3.1. Fungal Isolates
To study the morphological characterization, the samples were collected from decline affected citrus trees from representative orchards of tehsil and district
Sargodha and tehsil Bhalwal of Sargodha. For mango, the orchards of districts
Multan & Rahim Yar Khan and for guava tehsil Pattoki of district Kasur and tehsil
Sharaqpur of Sheikhupura. The areas were selected as representatives, based on their commodity based popularity as well as number of orchards. The samples were taken from twigs, branches and stem at collar region. The cultures were purified by sub culturing and finally 30 isolates were selected out of 300 samples based on their hosts used in the present study. The methodology for isolation and identification has already been mentioned in chapter 3.
6.3.2. Morphological Characterization
For morphological characterization, each isolate was maintained on Malt
Extract Agaer (MEA) medium. A 5 mm mycelial plug was taken from fresh growing culture of the fungi N. mangiferae and B. theobromae and aseptically placed in the center of petridish (90 mm). After 5 days at 25oC, morphological characters like colony color and patterns were studied. However, the conidial characters and measurements were recorded on conidial appearance.
The measurement of conidia, perithecia and other target structure was done using method reported by Fateh et al., 2006.
6.4.RESULTS 162
The morphological variability of the most common decline causing fungi N. mangiferae was studied based on hosts i.e. citrus mango and guava, plant parts i.e. branch and twig and location. The results are shown in the tables 6.1 to 6.6.
The isolates for decline affected citrus trees collected from tehsil and district Sargodha showedmorphologically similar characteristics irrespective of the plant part and location. The isolates on malt extract agar medium showed dark grey mycelial and produced appressed colonies five days after inoculation. However, there was a slight difference in the measurement of chain of arthrospores which were somewhat elongated (Fig.6.1), hyaline aseptate, and fusoid becoming 2-3 septate with central cell dark brown (Table 6.1).
In tehsil Bhalwal of Sargodha, isolates had exactly similar characteristics with one another. They even did not show slight change in the measurement of arthrospores. They also showed resemblance to the isolates taken from tehsil and district Sargodha (Table 6.2).
In case of mango, the isolates of N. mangiferae showed major difference in the mycelial pattern in the 90 mm petri plates. The mycelia started to rise instead of running in the appressed form. Mycelial color in case of mango after 5 days of inoculation turned creamy grey which was completely different fromN. mangiferae isolates taken from decline affected citrus trees. The arthrospores were also not much elongated continuously and mostly they were round shaped in the chain
(Table 6.3). The isolates from district Rahim Yar Khan were also similar to the isolates taken from district Multan (Table 6.4).
The isolates taken from decline affected guava trees show more closeness towards the mango isolates. There has been mixture of elongated and roundish 163
Table 6.1: Morphological characteristics of N. mangiferae isolates from citrus in
tehsil and district Sargodha.
Isolate Tree/ Mycelial Mycelial Arthrospores Description of Plant color pattern measurement arthrospores Part (after 5 (LxW) µm days) SW-6 Citrus/ Dark grey Appressed 5-12.5x2.5-10 Somewhat elongated Branch hyaline aseptate, fusoid becoming 2-3 septate with central cell dark brown SW-7 -ditto- -ditto- -do- 5-12.0x2.5-10 -ditto- SW-8 -ditto- -ditto- -do- 4.5-12.5x2.5-9 -ditto- SW-9 -ditto- -ditto- -do- 5-12.5x2.5-10 -ditto- SW-10 -ditto- -ditto- -do- 5-12.0x2.5-9 -ditto- Sar-6 Citrus/ -ditto- -do- 4.5-12.0x3.0- -ditto- Twigs 10 Sar-7 -do- -ditto- -do- 5-12.0x2.5-10 -ditto- Sar-8 -do- -ditto- -do- 5-12.5x2.5-10 -ditto- Sar-9 -do- -ditto- -do- 5-12.0x2.5-9 -ditto- Sar-10 -do- -ditto- -do- 4.5-12.0x3.0- -ditto- 10
Table 6.2: Morphological characteristics of N. mangiferae isolates from citrus in
tehsil Bhalwal, district Sargodha.
Isolate Tree/ Mycelial Mycelial Arthrospores Description of Plant color pattern measurement arthrospores Part (after 5 (LxW) µm days) BE-10 -ditto- Dark grey Appressed 5-12.0x2.5-10.5 elongated hyaline aseptate, fusoid becoming 2-3 septate with central cell dark brown BW-6 Citrus/ -ditto- -ditto- 5-12.0x2.5-10 -ditto- Twigs BW-7 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -ditto- BW-8 -ditto- -ditto- -ditto- 5-12.0x2.5-9 -ditto- BW-9 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -ditto- BW- -ditto- -ditto- -ditto- 5-12.0x2.5-10.5 -ditto- 10 164
Table 6.3: Morphological characteristics of N. mangiferae isolates from mango in
district Multan.
Isolate Tree/ Mycelial Mycelial Arthrospores Description of Plant color pattern measurement arthrospores Part (after 5 (LxW) µm days) BBR-6 Twigs Creamy Raised 5-12.0x2.5-10 Hyaline aseptate, grey fusoid becoming 2-3 septate with central cell dark brown
BBR-7 -ditto- -ditto- -ditto- 4.9-12.0x2.5-10 -ditto- BBR-8 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto- MKR- -ditto- -ditto- -ditto- 5-12.0x3.0-10 -ditto- 8 MKR- Branch -ditto- -ditto- 4.4-12.5x2.5-10 -ditto- 9 MKR- -ditto- -ditto- -ditto- 5.2-12.0x2.5-10 -ditto- 10 MSR- -ditto- -ditto- -ditto- 4.5-12.0x2.5-10 -ditto- 6 MSR- -ditto- -ditto- -ditto- 4.2-12.0x2.5-10 -ditto- 7 MSR- -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto- 8 MSR- -ditto- -ditto- -ditto- 5-12.0x2.5-10 -ditto- 9
165
Table 6.4: Morphological characteristics of N. mangiferae isolates from mango in
district Rahim Yar Khan.
Isolate Tree/ Mycelial Mycelial Arthrospores Description of Plant color (after pattern measurement arthrospores Part 5 days) (LxW) µm
LP-7 Twigs Creamy Raised 4.2-12.5x2.1-10 Hyaline aseptate, grey fusoid becoming 2-3 septate with central cell dark brown
LP-8 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto- LP-9 -ditto- -ditto- -ditto- 4.2-12.5x2.5-10 -ditto- KP-6 Branch -ditto- -ditto- 5.1-12.5x2.5-10 -ditto- KP-7 -ditto- -ditto- -ditto- 5-12.0x2.5-10 -ditto- KP-8 -ditto- -ditto- -ditto- 5.2-12.0x2.7-10 -ditto- MQ-6 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto- MQ-7 -ditto- -ditto- -ditto- 4.8-12.0x2.7-10 -ditto- MQ-8 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto-
Table 6.5: Morphological characteristics of N. mangiferae isolates from guava in
tehsil Pattoki of district Kasur.
Isolate Tree/ Mycelial Mycelial Arthrospores Description of Plant color (after pattern measurement arthrospores Part 5 days) (LxW) µm
PI-12 Twigs Creamy Raised 4.5-12.5x2.0-10 Hyaline aseptate, grey fusoid becoming 2-3 septate with central cell dark brown
PI-13 -ditto- -ditto- -ditto- 4.0-12.0x2.0-10 -ditto- PI-14 -ditto- -ditto- -ditto- 4.8-10.5x2.5-10 -ditto- PI-15 -ditto- -ditto- -ditto- 4.0-12.0x2.0-10 -ditto- PI-16 Branch -ditto- -ditto- 4.5-12.0x2.5-10 -ditto- PI-17 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -ditto- PI-18 -ditto- -ditto- -ditto- 4.5-12.5x2.8-10 -ditto- PI-19 -ditto- -ditto- -ditto- 5.2-12.0x2.4-10 -ditto- PI-20 -ditto- -ditto- -ditto- 5-12.5x2.5-10.5 -ditto- 166
Table 6.6:Morphological characteristics of N. mangiferae isolates from guava in
tehsil Sharaqpur of district Sheikhupura.
Isolate Tree/ Mycelial Mycelial Arthrospores Description of Plant color (after pattern measurement arthrospores Part 5 days) (LxW) µm
SP-11 Twigs Creamy Raised 5-12.5x2.5-10 Hyaline aseptate, grey fusoid becoming 2-3 septate with central cell dark brown SP-12 -ditto- -ditto- -ditto- 4.5-12.0x2.5-9 -do- SP-13 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -do- SP-14 -ditto- -ditto- -ditto- 5-12.5x3.0-10 -do- SP-15 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -do- SP-16 Branch -ditto- -ditto- 4.5-12.0x2.5-10 -do- SP-17 -ditto- -ditto- -ditto- 4.0-12.5x2.5- -do- 10.2 SP-18 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -do- SP-19 -ditto- -ditto- -ditto- 5-12.0x2.5-10.2 -do- SP-20 -ditto- -ditto- -ditto- 4.5-12.0x2.5-10 -do-
167
Fig.6.1:N. mangiferae arthrospores from citrus
Fig.6.2:N. mangiferae arthrospores from mango
168
arthrospores (Fig. 6.2). However, there has been no difference in mycelial colours and pattern when compared with mango isolates.
6.5. DISCUSSION
The morphological characteristics of the fungus described in current study were in agreement with those reported by Wilson (1949); Calavan and Wallace
(1954); Sutton and Dyko (1989) and El Atta and Nori (2014). The similarity between isolates of mango and guava shows that there has been a shift of attack of
N. mangiferae either from mango to guava or otherwise. The chances are that these are the same fungal isolates which attack both trees as both are grown in tropical climate and as discussed earlier that sometimes guava is intercropped in mango orchards and hence there has been a transfer of the fungus from mango to guava.
It has been found during the pathogenicity tests that the symptoms produced by N. mangiferae in both trees were almost similar, which is again a confirmation of their common origin (Fateh et al., 2016). In case of citrus, the morphology of N. mangiferae was a little bit different. One of the reasons might be the difference in climate where citrus is grown and secondly, it can be due to the fact that in citrus the fungus has been reported to produce branch wilt with cracks in a particular check pattern, while in mango and guava they either cause branch dieback, branch canker or twig die back (Mirzae et al., 2002; Saaiman, 1997).
However, the morphology of N. mangiferae isolates from all citrus, mango and guava had close resemblance with madrone canker isolates in USA. Madrone isolates of N. mangiferae are characterized as hyaline, aseptate, guttulate conidia becoming 1- to 3-septate, verisicolored or brown. Conidia had thin-walls, mostly fusoid but occasionally oblong or clavate with truncate bases. The isolates differed 169
in conidial length, width as well as in arthrospore size. Although there were statistically significant differences, there does not appear to be a geographical pattern in spore size or shape. The researchers in US conclude that N. mangiferae isolated from Pacific madrone belonged in the teleomorph genus Botryosphaeria and hence, diseases caused by this fungus can be managed using methods developed for other Botryosphaeria pathogens, such as B. dothidea(Elliott and
Edmonds, 2003).
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Chapter 7
FACTORS FAVORING THE DECLINE DISEASE AND DEVELOPMENT OF STRATEGIES FOR ITS MANAGEMENT 7.1. INTRODUCTION
Tree decline have been a universal problem and none of the forest, fruit and ornamental trees are safe from the disease. Mostly decline in trees has very complex etiology and is attributed to pathogens, various native organisms, climatic factors and agricultural or urban pollution. Where particular biotic or abiotic factors could not be singled out, they have been regarded as predisposing, inciting or contributing factors in „diseases of complex etiology‟. For example ongoing monitoring of eucalypt decline during recent droughts in eastern Australia, together with extensive one-time observations across temperate Australia, provided opportunities to further examine some hypotheses of decline and dieback that were largely based on retrospective investigations (Jurskis, 2005). Dieback in intervals can be distinguished from the process of chronic decline. Repeated dieback was associated with natural climatic extremes whereas chronic decline was associated with human management. Decline of forests in nature was associated with exclusion of fire and grazing, while decline of rural trees was mostly associated with pastures development (Fensham, 1998). Trees growing low in the landscape on soils with poor drainage and aeration were especially predisposed to decline. It appears that chronic abiotic stress causes tree decline when the function of roots is impaired by changes in soils. Climatic extremes can accelerate severe declines associated with human management. A variety of pests, „pathogens‟ and parasites can take advantage of trees that are stressed by environmental changes, especially eutrophication. Similarities between diebacks and declines in the Atlantic and 147 171
Pacific regions suggest a simple unifying concept of tree decline and dieback
(Karnosky, 2003).
A gradual decline in tree health is a common problem in urban and rural areas of Pakistan. Decline is not limited to certain specific trees but it is galloping towards almost all woody vegetation of forest, ornaments and fruits. Trees live long and over period of years they are invaded by number of insects, diseases and environmental effects. These trees are called stressed trees and often end up in decline and mortality (Bigler et al., 2004).
7.2. REVIEW OF LITERATURE
Pakistan has tropical and sub tropical climate in most of the fruit growing areas. Much information has been generated in general for the perennial crops in the tropics and sub tropics in the world. Diseases in the tropics may be complicated by interactions between different pathogens, or between pathogens and insect pests
(Holliday, 1980; Ploetz, 2006; Vandermeer et al., 2010; Anonymous, 2010).
Disease complexes involving a number of fungal pathogens or fungi and nematodes are common in tropical situations. Interactions between pathogens and environmental stress may also occur. Crops can become more susceptible to pathogen infections when weakened by environmental stresses such as drought, temperature extremes, and exposure to sunlight or wind (Agrios, 2005). Stressed plants, or plants sustaining damage caused by insects or other pathogens, may also be susceptible to attack by secondary pathogens or pathogens that infect through wounds. Nutrient deficiencies may increase the susceptibility of crops to disease. In tropical perennial crops, poor plant nutrition is likely to be a particularly important contributing factor to production losses (Palti, 1981). In addition to lower 172
production due to nutrient deficiency, low nutrition may predispose plants to diseases, increasing losses further. Nutrient deficiency causes the plant to become weakened and generally more susceptible to infection. Under such conditions, infection by weakly pathogenic species that would normally cause few problems may become more serious. The incidence and severity of particular diseases may also be linked to deficiencies of particular nutrients (Schroth et al., 2000).
Scientists agreed that the insect pests and diseases are not the only cause of tree decline but the tree age, environmental factors, tree nutrition, soil chemistry and above all the neglecting right production practices play a crucial role for decline of the trees. For example citrus decline in Pakistan is a complex or syndrome. As the phytoplasma, viruses, nematodes and fungi are involved in citrus decline but at the same time the production practices like tree density in the orchard, orchard sanitation, orchard irrigation and nutrition etc. are not accurate
(Arif et al. 2005; Burney et al., 2007). All these circumstances invite decline not only for citrus but also in other forest, ornamental and fruit trees (Bigler et al.,
2006).
Mango decline has been a problem for Pakistan since it was first time reported in 1995 at Muzaffar Garh. Lot of work has been done on mango decline in the past few years and the production technology is being altered to manage the issues (Fateh et al., 2006). Mango decline is a general term and it is used for different disorders in the mango trees which either slowly or rapidly decline the mango tree or eventually cause the death of tree
Guava decline is another big threat in Pakistan. As we have already limited area under cultivation and the stressed condition someday will let vanish this 173
precious fruit from lands of Pakistan. It is a national problem of Pakistan because since last decade the guava production has been declining. The problem of guava decline has been prevailing in Lahore, Sheikhupura, Faisalabad, Jhang and
Sargodha (Khushk et al., 2009).
Therefore, the present study was done to know the common factors favouring decline disorders especially in mango citrus and guava and on the basis of these factors listing the management strategies that can be adopted to avoid tree decline in future.
7.3. MATERIALS AND METHODS
7.3.1.Surveys for Factor Favoring Decline in Fruit Orchards
A proforma was designed to have survey in the citrus, mango and guava orchards randomly in their respective production areas as mentioned in table 7.1,
7.2 and 7.3 to record parameters such as tree crop, age of trese, variety, intercropping, method of irrigation, nutrition applied, pruning of trees, ploughing in the orchards and plant protection measures adopted to co relate this with disease index (%) found. The detail of the parameters was as under:
Tree Crop: Mango, citrus, guava
Age of the tree: Age of the tree was taken in the form of range. The trees were divided into different age groups i.e. less than 10 years, 10-15, 16-25, 26-50 and more than 50 years were represented by 3.
Variety: individual tree variety was taken into records
Intercropping: The field crop, other trees grown or otherwise no intercropping was observed
Irrigation: Source method and time of irrigation was observed 174
Nutrition: Kind of nutrients applied or not applied
Ploughing under tree canopy: Ploughing in the orchard especially under tree canopy was present or absent.
Plant protection measures: If any plant protection measures are adopted?
Disease Incidence in the Orchard and average severity: Incidence of the disease was recorded as the % age of affected plants in an observed population. While, the severity was recorded based on the area of infected individual tree.Factors were correlated with disease index (%).
7.3.2. Statistical Analysis
For the statistical analysis, SPSS software was used wherever needed. First of all the data was coded in binary form. Such kind of coding is needed where in data we have "Yes" or "No"entries. The data was imported from excel spread sheet to SPSS software. Finally the correlation was chosen for the analysis to see effect of each factor on decline disease index of citrus mango and guava.
7.4.RESULTS
According to table 7.1 survey conducted in 6 tehsils of district
Sargodha for observing the factors that can favor the decline disease in these areas has shown interesting results. In Tehsil Bhalwal 8citrus orchards out of 13 locations fell in the age group of 16-25 while the rest 5 are in the age group of 10-
15. The sole variety of citrus cultivated in these orchards was "Kinnow". In all young orchards disease index either was low or zero. This is because the young 175
Table 7.1: Factor affecting disease index (%) in selected orchards of citrus in district Sargodha.
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Chak No. Canal/F P P (Lime Bhalwal 4 SB 16-25 Kinnow P lood (FYM) A P paste) 31 Chak No. Canal/F P P (Lime 7 ML 16-25 Kinnow P lood (FYM) A P paste) 32 Chak No. Canal/F P P (Lime 7SB 16-25 Kinnow P lood (FYM) A P paste) 40 Chak No. Canal/F P P (Lime 7 ASB 16-25 Kinnow P lood (FYM) A P paste) 37 Chak No. Canal/F P P (Lime 8NB 16-25 Kinnow P lood (FYM) A P paste) 33 Chak No. Canal/F P P (Lime 9 NB 10-15 Kinnow A lood (NPK) P A paste) 15 Chak No. Chak 9 Canal/F P P (Lime Lokri 16-25 Kinnow P lood (FYM) A P paste) 36 0 Chak No. Canal/F P P (Lime 10 NB 10-15 Kinnow A lood (NPK) P A paste) 176
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Chak No. Canal/F P P (Lime 13 NB 16-25 Kinnow A lood (FYM) P P paste) 31 Chak No. Canal/F P P (Lime 18 NB 16-25 Kinnow P lood (FYM) A P paste) 37 Chak No. Canal/F P P (Lime 22 NB 10-15 Kinnow A lood *(NPK) P A paste) 0 Chak No. Canal/F P P (Lime 23 NB 10-15 Kinnow A lood (FYM) A P paste) 17 Chak No. Canal/F P P (Lime 26 NB 10-15 Kinnow A lood (NPK) P A paste) 0 Sargodha Chak No. Canal/F P P (Lime 24 SB 16-25 Kinnow A lood (FYM) A P paste) 20 Chak No Canal/F P P (Lime 27 SB 16-25 Kinnow P lood (FYM) A P paste) 37 Chak No Canal/F P P (Lime 28 SB 16-25 Kinnow A lood (FYM) P P paste) 20 Chak No Canal/F P P (Lime 30 NB 16-25 Kinnow P lood (FYM) A P paste) 27 Chak No Canal/F P P (Lime 48 NB 16-25 Kinnow P lood (FYM) A P paste) 29 177
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Chak No Canal/F P P (Lime 53 SB 16-25 Kinnow P lood (FYM) A P paste) 31 Chak No Canal/F P P (Lime 56 NB 16-25 Kinnow P lood (FYM) A P paste) 27 Chak No Canal/F P P (Lime 90 NB 16-25 Kinnow P lood (FYM) A P paste) 32 Chak No Canal/F P P (Lime 91 NB 16-25 Kinnow A lood (FYM) A P paste) 24 Chak No Canal/F P P (Lime 93 SB 16-25 Kinnow P lood (FYM) A P paste) 40 Chak No Canal/F P P (Lime 94 NB 16-25 Kinnow P lood (FYM) A P paste) 37 Chak No Canal/F P P (Lime 95 NB 16-25 Kinnow P lood (FYM) A P paste) 33 Chak No Canal/F P P (Lime 95SB 16-25 Kinnow P lood (FYM) A P paste) 32 Chak No Canal/F P P (Lime 101 SB 16-25 Kinnow A lood (FYM) P P paste) 24 Chak No Canal/F P P (Lime 112 NB 16-25 Kinnow P lood (FYM) A P paste) 29 178
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Chak No Canal/F P P (Lime 115 SB 16-25 Kinnow P lood (FYM) A P paste) 32 Chak No Canal/F P P (Lime 122 SB 16-25 Kinnow P lood (FYM) A P paste) 25 Sillanwali Chak No. Canal/F P P (Lime 118 NB 10-15 Kinnow A lood (NPK) P A paste) 15 Chak No. Canal/F P P (Lime 119 SB 16-25 Kinnow A lood (NPK) P P paste) 24 Chak No. Canal/F P P (Lime 120SB 10-15 Kinnow A lood (NPK) P A paste) 0 Chak No. Canal/F P P (Lime 122 NB 10-15 Kinnow A lood (NPK) P A paste) 12 Chak No. Canal/F P P (Lime 123 SB 16-25 Kinnow P lood (FYM) P P paste) 32 Chak No. 10-15 Canal/F P P (Lime 124 SB Kinnow A lood (NPK) P A paste) 0 Chak No. 10-15 Canal/F P P (Lime 127 SB Kinnow A lood (NPK) P A paste) 4 Chak No. 10-15 Canal/F P P (Lime 127 NB Kinnow P lood (NPK) P A paste) 15 179
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Chak No. 10-15 Canal/F P P (Lime 137 SB Kinnow A lood (NPK) P A paste) 0 Chak No. 10-15 Canal/F P P (Lime 147 NB Kinnow A lood (NPK) P A paste) 0 Chak No. 10-15 Canal/F P P (Lime 148 NB Kinnow A lood (NPK) P A paste) 12 Shahpur Canal/F P P (Lime Malakwal 16-25 Kinnow P lood (NPK) P A paste) 19 Canal/F P P (Lime Wadhi 16-25 Kinnow P lood (FYM) P P paste) 35 Kot Canal/F P P (Lime Maghrib 16-25 Kinnow P lood (FYM) P P paste) 20 Shahpur Canal/F P P (Lime Saddar 16-25 Kinnow P lood (FYM) A P paste) 27 Noor Canal/F P P (Lime Kallu 16-25 Kinnow P lood (FYM) A P paste) 27 Kandaan Canal/F P P (Lime Kalan 16-25 Kinnow P lood (FYM) A P paste) 31 Canal/F P P (Lime Jhavarian 16-25 Kinnow P lood (FYM) A P paste) 28 180
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Hussain Canal/F P P (Lime Shah 16-25 Kinnow P lood (FYM) P P paste) 25 Canal/F P P (Lime Kudyana 16-25 Kinnow P lood (FYM) P P paste) 23 Allahdad Canal/F P P (Lime Wala 16-25 Kinnow P lood (FYM) A P paste) 25 Chachar Canal/F P P (Lime Sharf 16-25 Kinnow P lood (FYM) A P paste) 28 Chak 128 Canal/F P P (Lime NB 16-25 Kinnow P lood (FYM) A P paste) 33 Sahiwal Kot Canal/F P P (Lime Pehalwan 16-25 Kinnow P lood (FYM) A P paste) 28 Biral Canal/F P P (Lime Sharif 16-25 Kinnow P lood (FYM) A P paste) 25 Canal/F P P (Lime Nawabpur 16-25 Kinnow P lood (FYM) A P paste) 24 Canal/F P P (Lime Vijh 16-25 Kinnow P lood (FYM) A P paste) 28 Canal/F P P (Lime Nehang 10-15 Kinnow A lood (FYM) P A paste) 0 181
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Canal/F P P (Lime Chohal 16-25 Kinnow P lood (FYM) A P paste) 23 Tirkhanwa Canal/F P P (Lime la 10-15 Kinnow A lood (FYM) P A paste) 0 Haveli Canal/F P P (Lime Majuka 16-25 Kinnow P lood (FYM) A P paste) 32 Pindi Canal/F P P (Lime Wala 16-25 Kinnow P lood (FYM) A P paste) 24 Canal/F P P (Lime Sial Sharif 16-25 Kinnow P lood (FYM) A P paste) 31 10-15 Canal/F P P (Lime Sangoraka Kinnow P lood (FYM) P A paste) 12 10-15 Canal/F P P (Lime Farooka Kinnow P lood (FYM) P A paste) 16 Sial Canal/F P P (Lime Dholka 16-25 Kinnow P lood (FYM) A P paste) 23 Muhamma Canal/F P P (Lime d Wala 16-25 Kinnow P lood (FYM) A P paste) 24 Jahane Canal/F P P (Lime Wala 10-15 Kinnow A lood (NPK) P A paste) 0 182
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Canal/F P P (Lime Dherowal 16-25 Kinnow P lood (FYM) A P paste) 28 Canal/F P P (Lime Chatror 16-25 Kinnow P lood (FYM) A P paste) 29 10-15 Canal/F P P (Lime Radhan Kinnow A lood (NPK) P A paste) 0 Kot Momin Chak No. 10-15 Canal/F P P (Lime 9 SB Kinnow A lood (NPK) P A paste) 0 10-15 Canal/F P P (Lime 19 SB Kinnow A lood (NPK) P A paste) 0 Canal/F P P (Lime 20 SB 16-25 Kinnow A lood (NPK) P A paste) 12 Canal/F P P (Lime 21 SB 16-25 Kinnow P lood (FYM) P P paste) 19 Canal/F P P (Lime 65 SB 16-25 Kinnow P lood (FYM) P P paste) 16 Canal/F P P (Lime 66 SB 16-25 Kinnow P lood (FYM) P P paste) 23 Canal/F P P (Lime Rawan 10-15 Kinnow A lood (NPK) P A paste) 0 183
Tehsil Location Age Variety Intercr Irrigati Nutriti Prunin Plough Plant Disease Group op on on g (P/A) ing Protecti Index (%) (P/A) (T/C), (P/A) under on (P/A) (HE/ tree If P Fl) canopy (which) (P/A) Dera Thoye Canal/F P P (Lime Wala 16-25 Kinnow P lood (FYM) A P paste) 24 Ghulapur Canal/F P P (Lime Bangla 16-25 Kinnow P lood (FYM) A P paste) 29 10-15 Jalla Canal/F P P (Lime Makhdum Kinnow P lood (FYM) A P paste) 16 Takht 10-15 Canal/F P P (Lime Hazara Kinnow A lood (FYM) P A paste) 12 Naseerpur 10-15 Canal/F P P (Lime Kalan Kinnow A lood (FYM) P P paste) 17 Canal/F P P (Lime Mateela 16-25 Kinnow P lood (FYM) A P paste) 25 Canal/F P P (Lime Dodha 16-25 Kinnow P lood (FYM) A P paste) 28 Midh Canal/F P P (Lime Road 16-25 Kinnow P lood (FYM) P A paste) 16 FYM: Farmyard Manure; N: Nitrogen; P: Phosphorus; K: Potassium
184
orchards 10-15 years group following pruning, fertilizer regimes, without ploughing under tree canopy and plant protection spray regimes while from orchards of 16-25 age reciprocal observations were done. Almost similar trend was seen in other tehsils i.e. Sargodha, Shahpur and Sahiwal. However, best management of the orchards among all tehsils was observed in tehsil Kot Momin and Sillanwali. (Table 7.1).
The overall correlation of various factors with the citrus decline disease index (%) was analysed using SPSS softare. The results are shown in Table
7.2. A perfect correlation was observed regarding age group, intercropping and nutrition of the orchards with disease index percentage. One to one correlation gave the accurate picture. The results were significant regarding the correlation between the age groups and disease index %. It was observed that in young orchards disease was minimum or nil, while in other higher age groups it was vice versa. Therefore, at alpha 0.02 a perfect correlation was found using two tailed correlation test
(Table 7.3).
The results are significant when we observe correlation of intercropping
(Table 7.4), nutrition application (Table 7.6), pruning of trees (Table 7.7) and ploughing under the tree canopy (Table 7.8) at the 0.01 level (2-tailed). The mode of irrigation (Table 7.5) and plant protection measures adopted (Table 7.9) were almost constant, hence correlation could not develop.
It is obvious from the results that orchards need care and management to avoid decline, especially the snescence affects the physiology of trees and they cannot resist degradation of cells.On the other hand if the growers adopt best management practices like pruning of the trees for deadwood removal, aeration and 185
Table 7.2:Correlations of factors causing citrus decline disease index (%).
Ploughing Plant Irrigation under treeProtectio Intercrop (T/C), Nutrition Pruning canopy n (P/A) If Disease Age Group (P/A) (HE/Fl) (P/A) (P/A) (P/A) P (which) Index(%) Age Group Pearson 1 .687** .b .687** .621** -.835** .b .843** Correlation Sig. (2- .000 . .000 .000 .000 . .000 tailed) N 86 86 86 86 86 86 86 86 Intercrop (P/A) Pearson .687** 1 .b .663** .607** -.634** .b .714** Correlation Sig. (2- .000 . .000 .000 .000 . .000 tailed) N 86 86 86 86 86 86 86 86 Irrigation (T/C), Pearson .b .b .b .b .b .b .b .b (HE/Fl) Correlation Sig. (2- ...... tailed) N 86 86 86 86 86 86 86 86 Nutrition (P/A) Pearson .687** .663** .b 1 .624** -.805** .b .736** Correlation Sig. (2- .000 .000 . .000 .000 . .000 tailed) N 86 86 86 86 86 86 86 86 `Pruning (P/A) Pearson .621** .607** .b .624** 1 -.723** .b .718** Correlation 186
Ploughing Irrigatio under tree
Age Intercrop n (T/C), Nutrition Pruning canopy Group (P/A) (HE/Fl) (P/A) (P/A) (P/A) Sig. (2- .000 .000 . .000 .000 . .000 tailed) N 86 86 86 86 86 86 86 86 Ploughing under Pearson -.835** -.634** .b -.805** -.723** 1 .b -.849** tree canopy Correlation (P/A) Sig. (2- .000 .000 . .000 .000 . .000 tailed) N 86 86 86 86 86 86 86 86 Plant Protection Pearson .b .b .b .b .b .b .b .b (P/A) If P Correlation (which) Sig. (2- ...... tailed) N 86 86 86 86 86 86 86 86 Disease Pearson .843** .714** .b .736** .718** -.849** .b 1 Index(%) Correlation Sig. (2- .000 .000 . .000 .000 .000 . tailed) N 86 86 86 86 86 86 86 86 **. Correlation is significant at the 0.01 level (2-tailed). b. Cannot be computed because at least one of the variables is constant. 187
Table 7.3: Correlation between citrus tree age groups with disease index (%) Disease Age Group Index (%) Disease Index (%) Pearson Correlation 1 .843**
Sig. (2-tailed) 0.000
N 86 86 Age Group Pearson Correlation 0.116 1
Sig. (2-tailed) 0.000
N 86 86
**. Correlation is significant at the 0.01 level (2-tailed).
Table 7.4:Correlation between intercrop in citrus orchards with disease index (%)
Disease Intercrop Index (%) (P/A) Disease Index (%) Pearson Correlation 1 0.714**
Sig. (2-tailed) 0.000
N 86 86 Intercrop (P/A)Pearson Correlation 0.714** 1
Sig. (2-tailed) 0.000
N 86 86
**. Correlation is significant at the 0.01 level (2-tailed).
188
Table 7.5: Correlation between modes of irrigation in citrus orchards with disese
index (%)
Disease Index Irrigation (%) (T/C), (HE/FI) Disease Index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 86 86
Irrigation(T/C),(HE/FI) Pearson Correlation .a .a
Sig. (2-tailed) .
N 86 86
a. Cannot be computed because at least one of the variables is constant
Table7.6: Correlation between nutrition in citrus orchards with disese index (%).
Disease Index Nutrition (%) (P/A) Disease Index (%) Pearson Correlation 1 0.736**
Sig. (2-tailed) 0.000
N 86 86
Nutrition (P/A) 0.736** 1
Sig. (2-tailed) 0.000
N 86 86
a. Cannot be computed because at least one of the variable is constant
189
Table 7.7: Correlation between prunings in citrus orchards with disease index (%).
Disease index Pruning (%) (P/A) Disease Index (%) Pearson Correlation 1 0.718**
Sig. (2-tailed) 0.000
N 86 86
Pruning (P/ A) Pearson Correlation 0.718** 1
Sig. (2-tailed) 0.000
N 86 86
**. Correlation is significant at the 0.01 level (2-tailed).
Table 7.8: Correlation between ploughing under tree canopy in citrus orchards with
disease index (%).
Disease Index Ploughing (%) under Tree Canopy (P/A) Disease Index (%) Pearson Correlation 1 -0.849**
Sig. (2-tailed) 0.000
N 86 86
Plouging under tree canopy (P/A) Pearson -0.849** 1 Correlation 0.000 Sig. (2-tailed)
N 86 86 **. Correlation is significant at the 0.01 level (2-tailed).
190
Table 7.9: Correlation between plant protection in citrus orchards with disease
index (%).
Disease Index Plant (%) Protection (P/A) Disease index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 86 86
Plant Protection (P/A) Pearson Correlation .a .a
Sig. (2-tailed) .
N 86 86
a. Cannot be computed because at least one of the variables is constant
191
Table 7.10:Factors affecting decline disease index in selected orchards of mango growing districts in Punjab.
District Tehsil Location Age Variety Interc Irrigation Nutrition Pruning Ploughing Plant Disease Group rop (T/C), (P/A) (P/A) under tree Protecti Index (P/A) (HE/Fl) canopy on (P/A) (%) (P/A) If P (which) Khane Kabir Qadirpur 26-50 Chaunsa P Canal/ P (FYM) A P A 39 wal wala Rawan Flood Canal/ 5 Kassi 26-50 Chaunsa P Flood P (FYM) A P A 39 Canal/ Solgi 26-50 Chaunsa P Flood P (FYM) A P A 41 Canal/ Matti Tal 26-50 Chaunsa P Flood P (FYM) A P A 51 Abbas Canal/ Pur 26-50 Chaunsa P Flood P (FYM) A P A 40 Canal/ 8 Kassi 26-50 Chaunsa P Flood P (FYM) A P A 47 Maula Canal/ Pur 26-50 Chaunsa P Flood P (FYM) A P A 49 Bilawal Canal/ Pur 26-50 Dosehri P Flood P (FYM) A P A 57 Basti Toheed Canal/ Nagar 26-50 Chaunsa P Flood P (FYM) A P A 65 Hassan Canal/ Pur 26-50 Desi P Flood P (FYM) P P P (Imida) 37 192
District Tehsil Location Age Variety Inter Irrigation Nutrition Pruning Ploughing Plant Disease Group crop (T/C), (P/A) (P/A) under tree Protecti Index (P/A) (HE/Fl) canopy on (P/A) (%) (P/A) If P (which) Multan P Multa Qasim Canal/ (Lambda, n Bela 16-25 Desi P Flood P (NPK) P A CoCl) 13 P Canal/ (Boadeau Nandla 16-25 Desi P Flood P (FYM) P P x paste) 31 Chah Nizam Canal/ Wala, 26-50 Desi P Flood P (FYM) A P A 52 Basti P Band Canal/ (Boadeau Bosan 26-50 Dosehri P Flood P (FYM) P P x paste) 33 Shuja Basti P bad Khokhra Canal/ (Boadeau n 26-50 Chaunsa P Flood P (FYM) P P x paste) 32 Shahpur Canal/ Ubbha 26-50 Chaunsa P Flood P (FYM) P P A 40 Abbas Canal/ Pura 26-50 Chaunsa P Flood P (FYM) P P A 40 Jalalp ur Pirwal Canal/ a Ghazipur 26-50 Chaunsa P Flood P (FYM) A P A 52 193
District Tehsil Location Age Variety Interc Irrigation Nutrition Pruning Ploughing Plant Disease Group rop (T/C), (P/A) (P/A) under tree Protecti Index (P/A) (HE/Fl) canopy on (P/A) (%) (P/A) If P (which) Manik Canal/ Wali 26-50 Desi P Flood P (FYM) P P A 39 Muzaff Muzaf Makhan ar Garh far Bela Canal/ Garh 26-50 Desi P Flood P (FYM) A P A 44 Rohillan Canal/ wali 26-50 Desi P Flood P (NPK) P P P (Imida) 27 Shah Canal/ Jamali 26-50 Desi P Flood P (FYM) A P A 44 Kot Ali Wala Canal/ Addu 16-25 Chaunsa P Flood P (FYM) P P A 29 Musay P Wala (Lambda, Tota Canal/ CoCl, 16-25 Pari P Flood P (NPK) P A Nativo) 13 Shuhrat Canal/ Wala 26-50 Desi P Flood P (FYM) A P P 40 Basti Canal/ Drigh 26-50 Desi P Flood P (FYM) A P P 52 Alipur Murad P pur Pull Canal/ (Boadeau 26-50 Chaunsa P Flood P (FYM) P P x paste) 31 Basti Canal/Floo kray 26-50 Chaunsa P d P (FYM) A P A 52 194
District Tehsil Location Age Variety Interc Irrigation Nutrition Pruning Ploughing Plant Disease Group rop (T/C), (P/A) (P/A) under tree Protecti Index (P/A) (HE/Fl) canopy on (P/A) (%) (P/A) If P (which) Basti Jat Canal/ Lashari 26-50 Desi P Flood P (FYM) A P A 41 Rahim Rahim Taranda Yar Yar Muham Khan Khan mad Canal/ Panah 26-50 Chaunsa P Flood P (FYM) A P A 32 Wahi Shah Muham Canal/ mad 26-50 Chaunsa P Flood P (FYM) A P A 52 Mianwali P Qureshia Canal (Boadeau n 26-50 Chaunsa P /Flood P (FYM) A P x paste) 32 Sadiq Ahmad abad Pur Canal/ Lumma 26-50 Desi P Flood P (FYM) A P A 41 Khan Chak 2P Canal/ Pur 26-50 Desi P Flood P (FYM) A P A 48 Chak 3 P Canal/ 26-50 Desi P Flood P (FYM) A P A 44 Liaqat Chak 22 Canal/ Pur A 26-50 Desi P Flood P (FYM) A P A 45 Chak 23 Canal/Floo 26-50 Desi P d P (FYM) A P A 49 195
District Tehsil Location Age Variety Interc Irrigation Nutrition Pruning Ploughing Plant Disease Group rop (T/C), (P/A) (P/A) under tree Protecti Index (P/A) (HE/Fl) canopy on (P/A) (%) (P/A) If P (which) Islam Canal/ Nagar 26-50 Desi P Flood P (FYM) A P A 48 Bahawa Baha Khanqah lpur walpu Sharif More Canal/ r than 50 Dosehri P Flood P (FYM) A P A 53 Chak 13 More Canal/ BC than 50 Chaunsa P Flood P (FYM) A P A 52 Chak 23 More Canal/ BC than 50 Chaunsa P Flood P (FYM) A P A 76 Nowsher Canal/ a 26-50 Chaunsa P Flood P (FYM) A P A 49 Munshi Canal/ Wala 26-50 Chaunsa P Flood P (FYM) A P A 57 Ahme Channi Pur Goth Canal/ East 26-50 Chaunsa P Flood P (FYM) P P A 32 Mehrab Canal/ Wala, 26-50 Dosehri P Flood P (FYM) A P A 60 Muhabba Canal/ t Pur, 26-50 Dosehri P Flood P (FYM) A P A 48 Basti Khokhra Canal/ n, 26-50 Langra P Flood P (FYM) A P A 45 196
Basti Canal/ Johnan 26-50 Langra P Flood P (FYM) A P A 61 FYM: Farmyard Manure; N: Nitrogen; P: Phosphorus; K: Potassium 197
reducing height, they can keep trees young and energeting for a long time. This can also be supplemented by using required nutrition after soil and water testing.
The results also showed that there is potential if we bring change in the methods of irrigation which is currently flooding, in which most water is wasted. If the growers adopt high efficiency irrigation such as drip, furrow or sprinkler irrigation this might reduce decline of trees further. Similarly the crop protection measures are almost uniform in all tehsils. If the growers start measures after proper identification of pests and diseases, they can add in health of trees and orchards.
To study factors favouring mango decline, a survey was done in the district of Khanewal, Multan, Muzaffar Garh, Rahim Yar Khan and Bahawalpur. In
Kabirwala tehsil of Khanewal almost at all locations mango orchard trees fell into age group of 26-50 years. At8locations chaunsa variety while in others locations desi and dosehri variety of mango was cultivated. Intercropping was common in all locations with wheat and cotton crop. At all of the locations and even the entire districts observed canal water was used as irrigation through flooding in mango orchards. All growers used farm yard manure as nutrition in the areas surveyed at
Kabirwala. Pruning was seen only at one location and ploughing under tree canopy was a common practice. No plant protection measures adopted except in one location where growers mostly spray Imidacloprid without knowing the need of chemical. Amost similar situation was found in district Multan except at 2 locations i.e. Qasim Bela and Nandla where orchards were relatively young and growers followed good agricultural practices and disease index (%) was found low.
198
Table 7.11:Correlations of factors causing mango decline disease index (%).
Age Age Group Intercrop Irri_mode Nutrition Pruning Ploughing under tree canopy Plant Protection Disease Index (%) Age Group Pearson Correlation 1 .a .a .a -.089 .030 -.068 .116 Sig. (2-tailed) . . . .548 .837 .644 .433 N 48 48 48 48 48 48 48 48 Intercrop Pearson Correlation .a .a .a .a .a .a .a .a Sig. (2-tailed) ...... N 48 48 48 48 48 48 48 48 Irri_mode Pearson Correlation .a .a .a .a .a .a .a .a Sig. (2-tailed) ...... N 48 48 48 48 48 48 48 48 Nutrition Pearson Correlation .a .a .a .a .a .a .a .a Sig. (2-tailed) ...... N 48 48 48 48 48 48 48 48 Pruning Pearson Correlation -.089 .a .a .a 1 -.342* .633** -.676** Sig. (2-tailed) .548 . . . .017 .000 .000 N 48 48 48 48 48 48 48 48 Ploughing under tree Pearson Correlation .030 .a .a .a -.342* 1 -.311* .542** canopy Sig. (2-tailed) .837 . . . .017 .032 .000
N 48 48 48 48 48 48 48 48 199
crop
Inter
Plant
canopy
Disease Disease
Pruning
Nutrition
Ploughing
Index (%)
Irri_mode
Protection
under tree under tree Group Age Plant Protection Pearson Correlation -.068 .a .a .a .633** -.311* 1 -.600** Sig. (2-tailed) .644 . . . .000 .032 .000 N 48 48 48 48 48 48 48 48 Disease Index (%) Pearson Correlation .116 .a .a .a -.676** .542** -.600** 1 Sig. (2-tailed) .433 . . . .000 .000 .000 N 48 48 48 48 48 48 48 48 *. Correlation is significant at the 0.05 level (2-tailed).
**. Correlation is significant at the 0.01 level (2-tailed). a. Cannot be computed because at least one of the variables is constant.
200
Table 7.12: Correlation between age agroup in mango orchards with disease index
(%).
Diseas index Age Group (%) Disease Index (%) Pearson Correlation 1 0.116
Sig. (2-tailed) 0.433
N 48 48
Age Group Pearson Correlation 0.116 1
Sig. (2-tailed) 0.433
N 48 48
Correlation is not significant at the 0.01 level (2-tailed).
Table 7.13: Correlation between itercropping in mango orchards with disease index
(%).
Diseas index Intercrop (%) Disease Index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 48 48
Intercropping Pearson Correlation .a .a
Sig. (2-tailed) .
N 48 48
a. Cannot be computed because at least one of the variable is constant
201
Table 7.14: Correlation between irrigation modes in mango orchards with disease
index (%).
Diseas index Irrigation (%) mode Disease Index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 48 48
Irrigation mode Pearson Correlation .a .a
Sig. (2-tailed) .
N 48 48
a. Cannot be computed because at least one of the variable is constant
Table 7.15: Correlation between nutrition in mango orchards with disease index
(%).
Diseas index Nutrition (%) Disease Index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 48 48
Nutrition Pearson Correlation .a .a
Sig. (2-tailed) .
N 48 48
a. Cannot be computed because at least one of the variable is constant
202
Table 7.16: Correlation between pruning of trees in mango orchards with disease
index (%).
Diseas index Pruning (%) Disease Index (%) Pearson Correlation 1 -0.676**
Sig. (2-tailed) 0.000
N 48 48
Pruning Pearson Correlation -0.676** 1
Sig. (2-tailed) 0.000
N 48 48
**. Correlation is significant at the 0.01 level (2-tailed)
Table 7.17: Correlation between ploughing under tree canopy in mango orchards
with disease index (%).
Diseas index Ploughing (%) under tree canopy Disease Index (%) Pearson Correlation 1 0.542**
Sig. (2-tailed) 0.000
N 48 48
Plowing under tree canopy Pearson Correlation 0.542** 1
Sig. (2-tailed) 0.000
N 48 48
**. Correlation is significant at the 0.01 level (2-tailed)
203
Table 7.18: Correlation between pant protection in mango orchards with disease
index (%).
Diseas index Plant (%) Protection Disease Index (%) Pearson Correlation 1 -0.600**
Sig. (2-tailed) 0.000
N 48 48
Plant Protection Pearson Correlation -0.600** 1
Sig. (2-tailed) 0.000
N 48 48
**. Correlation is significant at the 0.01 level (2-tailed)
204
Locations surveyed in Muzaffar Garh, Rahim Yar Khan and Bahawalpur had shown results on same pattern as that of Khanewal and Multan (Table 7.10).
The correlation through statistical analysis showed the significance of various factors with disease index (%) of mango decline (Table 7.11). The results were significant against the factors like pruning (Table 7.16), ploughing under the canopy of mango trees (Table 7.17) and the plant protection measures adopted
(Table 7.18) at the 0.01 level (2-tailed). Other factors like age group had shown no significance, as tree of all ages were victim of decline. The correlation of intercropping, irrigation and orchards nutritions was not found due to similarity in the practices.
According to table 7.19 for guava districts Kasur, Sheikhupura and
Nankana were surveyed. In district Kasur, trees age group of 10-15 and 16-25 with variety mix of Safeda, Allahabad and Karela. Intercropping of either wheat or berseem was observed. In all of the orchards irrigation source was canal and method was flooding. Proper nutrition was followed only at few places. Similar situations were found in district Sheikhupura and district Nankana.
The statistical correlation showed that the guava decline is significantly affected by age group, nutrition, pruning, ploughing under tree canopy and plant protection measures adopted or neglected in the guava orchards. (Table 7.21, 7.23,
7.25. 7.26 and 7.27). However, decline in guava is not as severe as it has been observed in mango.
The above results indicate that decline diseases can appear in any age group of the disease but with different disease index % ages. However, it is very clear that the young orchards are relatively healthy in all commodities and in all locations. 205
Table 7.19:Factors affecting decline disease index in selected orchards of guava growing districts in Punjab.
District Tehsil Location Age Variety Inte Irrigation Nutritio Prunin Plough Plant Disease Group rcrop (T/C), n (P/A) g (P/A) ing Protectio Index(% (P/A) (HE/Fl) under n (P/A) If ) tree P (which) canopy (P/A) Kasur Kasur Qaisar 16-25 Allahaba P Canal/Flood A A P A 17 Garh d Dolaywa 10-15 Allahaba P Canal/Flood A A P P (Lime 20 la d paste) Noor 16-25 Safeda P Canal/Flood A A P A 19 Shah Wali New City 10-15 Safeda P Canal/Flood A A P A 9 Kasur Sadar 10-15 Safeda P Canal/Flood P (FYM) P A P (Lime 23 Diwan paste) Chuniy Khara 16-25 Karela P Canal/Flood A A P A 8 an Kot 10-15 Karela P Canal/Flood P (FYM) A A P 23 Gurdas Bordeaux Wala Paste Pattoki Gehlan 16-25 Safeda P Canal/Flood A A P A 19 Pathak
206
District Tehsil Location Age Variety Inte Irrigation Nutritio Prunin Plough Plant Disease Group rcrop (T/C), n (P/A) g (P/A) ing Protectio Index(% (P/A) (HE/Fl) under n (P/A) If ) tree P (which) canopy (P/A) Rukan 16-25 Allahaba P Canal/Flood A A P P (Lime 24 Pura d paste) Sehjowal 26-50 Allahaba P Canal/Flood A A P A 33 d Sheikhupu Feroze Faizpur 26-50 Karela P Canal/Flood A A P A 16 ra wala interchan ge, Adda 10-15 Safeda P Canal/Flood A A A P (Lime 17 Thabal paste) Noor 10-15 Allahaba P Canal/Flood A A P P (Lime 28 Shah d paste) Burj 16-25 Allahaba P Canal/Flood A A P A 19 Attari d Saggian 16-25 Safeda P Canal/Flood A A P P (Lime 17 Khurd paste) Thikriwa 16-25 Safeda P Canal/Flood A A P P (Lime 8 la paste) Sharaq Sharaqpu 16-25 Safeda P Canal/Flood P (NPK) A A P 12 pur r (Bordeaux paste) 207
District Tehsil Location Age Variety Inte Irrigation Nutritio Prunin Plough Plant Disease Group rcrop (T/C), n (P/A) g (P/A) ing Protectio Index(% (P/A) (HE/Fl) under n (P/A) If ) tree P (which) canopy (P/A) Ghareeba 10-15 Karela P Canal/Flood A A P P (Lime 9 bad paste) Sukhanw 10-15 Karela P Canal/Flood P (NPK) P A P 15 ala (Bordeaux paste) Kot 10-15 Safeda P Canal/Flood A P P A 15 Mahmoo d Nankana Nankan Jalal 16-25 Safeda P Canal/Flood A P P A 9 Sahib a Sahib Nou Giller 10-15 Safeda P Canal/Flood P (FYM) P A P (Lime 8 Wala paste) 10-15 Safeda P Canal/Flood P (NPK) P P P 27 Magtan (Bordeaux Wala paste) Adda Pul 10-15 Safeda P Canal/Flood A A P A 19 Torian 10-15 Safeda P Canal/Flood A P P A 12
Chachka y Gill 208
District Tehsil Location Age Variety Inte Irrigation Nutritio Prunin Plough Plant Disease Group rcrop (T/C), n (P/A) g (P/A) ing Protectio Index(% (P/A) (HE/Fl) under n (P/A) If ) tree P (which) canopy (P/A) Mirza 10-15 Safeda P Canal/Flood A P A A 8 Pur Mandi 10-15 Safeda P Canal/Flood P (NPK) P P P (Lime 17 Faziabad paste)
FYM: Farmyard Manure; N: Nitrogen; P: Phosphorus; K: Potassium
209
Table 7.20: Correlation of factors causing guava decline with disease index
n
tree tree
Age Age
(%)
Plant
Index
under
Group
canopy
n mode n mode
Disease Disease
g
Pruning
Ploughin
Protectio
Irrigattio Nutrition
Intercrop Age Group Pearson Correlation 1 .a .a -.332 -.415* .318 -.383* .608** Sig. (2-tailed) . . .091 .031 .106 .049 .001 N 27 27 27 27 27 27 27 27 Intercrop Pearson Correlation .a .a .a .a .a .a .a .a Sig. (2-tailed) ......
N 27 27 27 27 27 27 27 27 Irri_mode Pearson Correlation .a .a .a .a .a .a .a .a Sig. (2-tailed) ......
N 27 27 27 27 27 27 27 27 Nutrition Pearson Correlation -.332 .a .a 1 .427* -.677** .350 -.684** Sig. (2-tailed) .091 . . .026 .000 .074 .000 N 27 27 27 27 27 27 27 27 Pruning Pearson Correlation -.415* .a .a .427* 1 -.299 .105 -.562** Sig. (2-tailed) .031 . . .026 .130 .603 .002 N 27 27 27 27 27 27 27 27 Ploughing Pearson Correlation .318 .a .a -.677** -.299 1 -.276 .594** under tree can. Sig. (2-tailed) .106 . . .000 .130 . .164 0.001 N 27 27 27 27 27 27 27 27 210
Age Group Intercrop Irrigation mode Nutrition Pruning Ploughing under tree canopy Plant Protection Disease Index (%) Plant Protection Pearson Correlation -.383* .a .a .350 .105 -.276 1 -.571** Sig. (2-tailed) .049 . . .074 .603 .164 .002 N 27 27 27 27 27 27 27 27 Disease Index Pearson Correlation .608** .a .a -.684** -.562** .594** -.571** 1 (%) Sig. (2-tailed) .001 . . .000 .002 .001 .002 N 27 27 27 27 27 27 27 27 *. Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed). a. Cannot be computed because at least one of the variables is constant.
Table 7.21: Correlation between age groups in guava orchards with disease index (%).
Diseas index Age Group (%) Disease Index (%) Pearson Correlation 1 0.608**
Sig. (2-tailed) 0.001
N 27 27
Age Group Pearson Correlation -0.608** 1
Sig. (2-tailed) 0.001
N 27 27
**. Correlation is significant at the 0.01 level (2-tailed)
Table 7.22: Correlation between intercropping of trees in guava orchards with disease
index (%).
Diseas index Intercrop (%) Disease Index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 27 27
Intercrop Pearson Correlation .a 1
Sig. (2-tailed) .
N 27 27
a. Cannot be computed because of the variables is constan
ii
Table 7.23: Correlation between irrigation modes in guava orchards with disease index
(%).
Diseas index Irrigation (%) mode Disease Index (%) Pearson Correlation 1 .a
Sig. (2-tailed) .
N 27 27
Irrigation mode Pearson Correlation .a .a
Sig. (2-tailed) .
N 27 27
a. Cannot be computed because of the variables is constan
Table 7.24: Correlation between nutrition applications in guava orchards with disease
index (%).
Diseas index Nutrition (%) Disease Index (%) Pearson Correlation 1 -0.684**
Sig. (2-tailed) 0.000
N 27 27
Nutrition Pearson Correlation -0.684** 1
Sig. (2-tailed) 0.000
N 27 27
**. Correlation is significant at the 0.01 level (2-tailed)
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Table 7.25: Correlation between pruning of trees in guava orchards with disease index
(%).
Diseas index Pruning (%) Disease Index (%) Pearson Correlation 1 -0.562**
Sig. (2-tailed) 0.002
N 27 27
Pruning Pearson Correlation -0.562** 1
Sig. (2-tailed) 0.002
N 27 27
**. Correlation is significant at the 0.02 level (2-tailed)
Table 7.26: Correlation between ploughing under tree canopy in guava orchards with
disease index (%).
Diseas index Ploughing (%) under tree canopy Disease Index (%) Pearson Correlation 1 0.594**
Sig. (2-tailed) 0.001
N 27 27
Age Group Pearson Correlation 0.594** 1
Sig. (2-tailed) 0.001
N 27 27
**. Correlation is significant at the 0.01 level (2-tailed)
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Table 7.27: Correlation between plant protection in guava orchards with disease index
(%).
Diseas index Plant (%) Protection Disease Index (%) Pearson Correlation 1 0.571**
Sig. (2-tailed) 0.002
N 27 27
Plant Protection Pearson Correlation 0.571** 1
Sig. (2-tailed) 0.002
N 27 27
**. Correlation is significant at the 0.02 level (2-tailed)
The senescence in tree has also greatly aggravated the problems.Especially in case of mango where the age group is either 26-50 years or even more, the disease index has touched 76% in Bahawalpur. However, in case of citrus and guava as the orchards were not too old the disease index was not much high.
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7.5. DISCUSSION
A significant correlation was observed in citrus for trees age and disease index.
It means that with an increase in tree age there was a corresponding increase in disease index.
With increasing age, the efficiency of tree for performing physiological function lowersi.e. lower photosynthetic rates, decreased growth rates, shifting of carbon resources to different parts of the plant and reductions in foliar efficiency, leaf size and gas exchange rates (Kaufmann, 1996; Ryan and Yoder, 1997; Carrer and
Urbinati, 2004; Martínez-Vilalta et al., 2007).Similarly, the larger tree size and the structural complexity associated with tree aging increase the maintenance respiration costs and reduce the efficiency of water transport; both tend to reduce growth (Weiner and Thomas, 2001; Carrer and Urbinati, 2004; Pennisi, 2005).
It has been reported that citrus productivity life span is only ten years, which is the prime age of production, whereas it is not uncommon for citrus orchards to remain in production for over 50 years in many other countries.It has also been mentioned that the average age of citrus tree in Pakistan is about 25 years. Therefore, the significant correlation between citrus age and disease index in the current study is supported by the previous findings of Ahmed et al.(2006) and Niaz et al. (2004). It has also been observed that fruit trees which are invaded by fungi in early age, react more severely in the advanced stage (Fraser and Singh, 1966).
In Pakistan more than 75% farmers are considered small landholders and even their holdings are shared. The small landholdings compel the growers for intensive cropping. Same is the case with citrus orchards like other fruit trees in Pakistan. v
vi
Therefore, intercropping of different kinds of fodders, cereals and other trees are a common practice in the country. Intercropping in the orchards is in a way beneficial for citrus growers, as it meets the needs of fodder for his livestock, grain for household use, fuel for his kitchen etc. At the same time the harmful pests and pathogen of diseases are harboured by these alternate hosts. This happens only when the intercropping is done with competing plants and unmatching life cycles. Same happened in trees under current study.
It was observed that rigorous and deep ploughing was done even under the canopy of trees which resulted in increased tree decline incidence as compared to orchards without intercropping. Intercropping also disturbs citrus orchards with unwanted spray and irrigation regimes received by the intercropped plants (Johnson,
2006). However, from rest of the world it has been reported that the intercropping of leguminous crops in citrus orchards help in the fertility of the orchards. Similarly if intercropped plants receive nitrogen, phosphorus and potassium fertilizers, indirectly these are also available to citrus trees as well as encourage the population of friendly insects. For example Sohail et al.(2013) reported that maizeintercropped plots of each citrus variety had low infestation of citrus miner and high population of coccinellids and Chrysoperla carnea when maize was intercropped in citrus orchards between the tree rows away from tree canopies.
In most of the surveyed orchards in citrus areas, nutrition was being provided either in the form of farmyard manure or as NPK. However, it was important to know that only about 18% of the areas surveyed were using the recommended doses of NPK fertilizers (700:350:700 g of NPK per tree with 4-6 kg FYM). Nutrient management in vi
vii
citrus influences flowering, fruit set, fruit size, the amount of vegetative growth and other plant characteristics. Nutrient management can help growers to have earlier, heavier fruit set (Ibrahim et al., 2004; Abd-Allah, 2006; Alva et al., 2006). Improper crop nutrition impairs plant genetic resistance to invasivepathogens, decreases yield and reduces productive life of the plant. It is interesting that in the surveyed areas where intercropping was practiced, NPK fertilizers were applied to the intercropped crops but growers did not apply fertlizers to citrus trees (Razi et al., 2011).
In case of mango, significant correlation was found in pruning, ploughing under the tree canopies and plant protection measures adopted. The large unpruned trees were mostly the victim of tree decline. Zero pruning in mango orchards has been reported in Pakistan to be a predisposing factor of mango decline (Naqvi and Perveen,
2015). Pruning is basically done to reduce tree size, to givie shape, removal of deadwood and upright branches as they are not productive, rather they help in hibernation of various insect pests and disease causing organisms. Pruning helps in increasing vigor of trees (Masood et al., 2014). It was seen that in Multan, where the pruning has been observed in quite a few orchards, the disease index percentage is relatively low as compared to other areas where it was not practiced. It is important to remember that only pruning without plant protection measures does not provide suitable results. This is because decline causing fungi are opportunistic in nature and enter through wounds. By pruning trees only they have a good chance to invade trees.
Hence, pruning coupled with plant protection measure come up with effective tree management (Al Adawi et al., 2006; Fateh et al., 2006; Fateh et al., 2009). The pruning and plant protection are integral for the health of mango trees (Poland et al., vii
viii
2006). Similarly, ploughing under tree canopy which injures roots provides an easy entry for the opportunistic fungi to invade mango trees and cause mortality. It has been observed especially, the fungus Ceratocystis fimbriata in mango has entered through root wounds (Saeed et al., 2007; Masoodet al., 2011).
In guava, a significant correlation was recorded for disease index with age group, nutrition, pruning, ploughing under tree canopy and plant protection measures.
As it has been discussed in citrus and mango, the age of tree is one of the biggest factors inviting the decline of trees, same is the case in guava as well (Khushk et al.,
2009). It has been discussed in details earlier in case of mango that how pruning and plant protection measures are linked. Pruning reduces the disease pressure and improves the vigor of guava trees (Tomita et al., 2016). The nutrition in guava orchards is seldom found especially the application of NPK is not adopted. However, nutrition is given in the form of farmyard manure. However, it has been reported that one percent increase in these factors results in 0.21 and .016 percent increase in guava yield (Ishtiaq et al., 2012). The major difference that has been observed in the severity of decline disease of guava from mango can be the difference in height as well as the density of leaves and branches. It was observed that guava trees were not as large as mango trees, having few branches which would allow the aeration and sunlight as well. However, tree injuries were common which were the entry point of various fungi as reported by Abbas et al.(2014). It is also worth mentioning to discuss here that
Pakistan is among the water deficient countries and cannot afford flood irrigation for its orchards or other crops. Secondly, the excessive standing water is lethal in two ways that it causes suffocation of trees as well as it can act as medium for spread of
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diseases from diseased trees to healthy ones. The country has been introduced to high efficiency irrigation systems and there is dire need to adopt them for conserving precious water as well as protect plants (Malik et al., 2004; Khushket al., 2009; Mirjat et al., 2011).
Based on the results of above study, the following short term management recommendations can be given:
1. Make sure to buy healthy planting material from reliable nurseries.
2. Pruning for having vigorous trees and providing quality fruits.
3. Follow recommendation of plant protection specialists for suitable and safe
spray regimes.
4. Analysis based nutrition for producing quality fruits may be adopted under
the guidance of nutrition specialists.
5. Wounding of the tree may be avoided to check entry of pests and
pathogens.
6. Intercrop with companion crops, such as legumes or with matching life
cycles.
7. Floor management may be encouraged, using local grass species without
disturbing the root zones of trees.
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Chapter 8
GENERAL DISCUSSION
8.1. ASSESSMENT OF DECLINE IN CITRUS, MANGO AND GUAVA
The decline disease assessment in citrus, mango and guava growing areas of
Punjab presents a gloomy picture for the respective industries. In all three commodities the decline has resulted from a number of biotic factors such as citrus tristeza virus (Arif et al., 2005); phytoplasma, bacteriae.g. Xanthomonas compestrisare involved in citrus decline (Burney et al., 2007). In Pakistan, the role of fungi in citrus decline has not been studied widely by scientists. However, the fungal involvement in citrus decline is a popular research topic in Oman, Iran and even in USA (Sutton and
Dyko, 1989; Mirzaee et al., 2003; Al Sadi et al., 2014). In Pakistan very limited research has been done to acknowledge the fungi in decline development. A list of fungi has been reported to cause decline symptoms. This IncludesLasiodiplodia hormozganensis, L. theobromae, Fusarium solani, Phytophthora sp., Neoscytalidium dimidiatum and Nattrassia mangiferaebeing the most common (Al Sadi et al., 2014;
Safdar etal., 2010; Fateh et al., 2016).
Besides biotic factors, the human manipulations regarding orchard management, floods, poorly drained soils, water logging, droughts, and climatic chnges push trees towards decline. It has been observed that poorly drained soils and deficiencies lead to die back (Chauhdary, 2003). Most of the orchards in Sargodha,
Bhalwal and Shahpur had old citrus plantations with intercropping of wheat, berseem and barley even under the canopy of trees, competing with feeder roots of trees in the
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198 xi
root zone.In the surveyed orchards where dead wood removal and pruning was not commonly practiced theorchards looked like forest(Ali et al., 2004).
The most available nutrition to the citrus trees was farmyard manure which at many places were dumped fresh in the orchards and caused burning of leaves. This practice can badly influence the soil pH and soil salinity. Imbalanced nutrition resulted in deficiency and toxicity of some elements e.g. zinc deficiency in the orchards which also promoted die back symptoms in trees. Although, it has been reported that citrus orchards are mostly macronutient deficient (Stenico et al., 2009). Another major issue is the quality of sub soil water which is unfit for irrigation and played an important role to destroy the citrus orchards. Canal water is not frequently available and flooding is done whenever it is available. It means there is no timely irrigation followed in the orchards.
Generally, more than 15% disease index of any disease at any location or any commodity is considered alarming. In district Sargodha overall disease index ranged from minimum 10.36 to maximum 29.41% which requires the immediate attention of the scientists and other stakeholders to have more research for management of the system through trainings and education (Fateh et al., 2017).
Among the insect pests, leaf miner, citrus psylla, whitefly and lemon butterfly were common. The gaps in the orchards were filled with poor quality planting material. So a complex of soil nutrients, insect pests and improper irrigation was observed which are all limiting factors for the growth and favorable for the disease
(Khan et al., 2011).
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In tehsils like Kot Momin and Sillanwali,the growers were following integrated orchard management approaches e.g. pruning of trees; plant protection sprays for insect pest and diseases on proper guidance from plant protection specialists; soil and water analysis for nutrition; orchard floor management without intercropping and unnecessary ploughing in the orchards. These good agricultural practices make
Sillanwali and Kot Momin better as compared to other citrus growing tehsils.
Moreover, nutritional study has been conducted in all tehsils of Sargodha and it was obvious that the above mentioned 2 tehsils were comparatively much better in nitrogen, potassium and phosphorus contents (Razi et al., 2011). Pruning in most of the orchards resulted in proper shapes of trees and no brancheswere touching the ground oroverlapping each other. Ploughing was not being done under the tree canopies and hence there was no chance of damage to the roots to provide openings to opportunistic fungi. Pruning of the trees reduces the pests and diseases and improves the equal flow of nutrients to whole tree making it vigorous to produce more quality food. It keeps the size of the tree in control and makes easy the field practices, sprays as well as fruit harvesting without much damage (Van Vuuren and Graca, 1996). In few orchards an effort was made to paste the lime and Bordeaux paste as well. This was enough to avoid the opportunistic fungi invading the stems as well as insects creeping into the bark cracks such as spider mite (Overmeer, 1985).
The localities surveyed in Bhalwal include Chak No. 10 /NB, Chak No. 10/1 were well managed. Moreover, the growers were literate and well aware of the orchard management. Most of them were posted on the influential posts as well as had links to the experts in citrus production technology. This may also be due to the international xii
xiii
projects running in the area. The trees were well pruned with good shapes having ages of 10-15years. Many orchards were having the lime pasting and some were sprayed with suitable pesticides. There was almost an equal ratio of farmers who were maintaining the orchards themselves as well as those whose orchards were maintained by the attendants. These orchards were being continuously monitored by the Global
Gap Certification Project of USAid. The experts guided the growers at each stage to perform different activities at different phenological stages (Anonymous, 2008).
Mango decline is also a problem of all traditional mango growing areas of
Pakistan. The survey to record the occurrence of mango decline was done in
Khanewal, Multan, Muzaffar Garh, Rahim Yar Khan and Bahawalpur distrcits which are famous for mango production. Maximum disease index of 53.30% was recorded in district Bahawalpur. During survey the large overlapping trees were observed in this district. Most of the areas surveyed had more than 100 years old plantations (Fateh et al., 2016). The growers were not even aware of the disease and they thought that senescens had resulted in such symptoms of the trees. Secondly, mango orchards are not given first importance rather the intercropped crops are considered important to the growers. The large farms are owned by the absent landlords who stay abroad and leave orchard management on their managers (munshies). These munshies strongly believe in traditional mango cultivation and show harshness towards the adoption of innovative technologies (Khushk et al., 1996).
The farmers have lost their interest in the mango orchards and taking more interest in the housing colonies. The orchards were neglected and animal grazing and lopping of trees were common. Most of the trees have been uprooted. The existing xiii
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trees had many wounds. The age of the trees ranged from 26-50 years. The old age itself a problem for the trees as they are more vulnerable to be attacked by the diseases
(Kazmi et al., 2005; Malik et al., 2005). However, mango tree decline was not limited to only old age trees but equally found on young trees as well. The trees were large in size, overlapping with each other giving a complete shade in the orchard and not allowing any penetration of sunlight. The pruning of the trees was like sin for the growers (Jiskani, 2002).
Flood irrigation was adopted in the orchards which could easily spread the disease from infected trees to healthy ones. The water was directly touching the stems of the trees and leaf drop was much more common. The chemical fungicides which were not supposed to be for mango were sprayed intentionally on the trees i.e. the chemicals left over after spraying on cotton and other crops (Iqbal et al.. 2007).
Along the Multan Khanewal and Multan Shujabad Road, many orchards were pruned and few orchards also showed the lime pasting around tree trunk that showed that these orchards are not as neglected as the orchards along Band Bosan Road.
Another reason may be that orchard along Shujabad road were in the area of Mango
Research Station Shujabad and had links with the expert. Therefore, the growers were taking interest in the management of their orchards and decline occurrence in these orchards was little bit less (Fateh et al., 2016).
Guava decline is another big threat in Pakistan. As we have already limited area for guava cultivation in Punjab and due to the stressed conditions someday this precious fruit will vanish from lands of Pakistan. It is a national problem of Pakistan because since last decade the guava production has been declining. The problem of xiv
xv
guava decline has been prevailing in Lahore, Sheikhupura, Faisalabad, Jhang and
Sargodha (Khushk et al., 2009). Maximum guava decline disease index was observed in Kasur (18%) followed by Sheikhupura (17%) and minimum of 14% in Tehsil
Nankana. Kasur being the nursery industry area, the nursery growers are interested in guava in high density for sale rather than in better management to earn from the fruit.
Most of the trees are not grown in proper rows, rather overlapping with each other in such a way that all the operation like sprays, pruning and machine operations are difficult. Improper cuttings and non treatment of wounds are giving rise to guava decline situation. However, proper guava cultivation has been observed in
Sheikhupura but with intercropping of wheat and berseem. Ploughing inside the orchards is increasing tree infection by making injuries. Nankana is comparatively low in disease as mostly there are new plantations with proper planning and better management strategy.
It is concluded that the situation of decline in citrus, mango and guava orchards is alarming and almost every orchard faced the problem. The situation warrants strict control strategies for its management. It is also emphasized that the farming community be trained in this regard to adopt better management strategies.
8.2. COMMON SYMPTOMS OF DECLINE IN CITRUS, MANGO AND
GUAVA
The common symptoms found in the orchards included the bark splitting, gummosis, leaf drooping and discoloration, twig blight, die back and the eventual tree mortality. In case of citrus decline, however, there are few additional symptoms like yellowing, dwarfing or bunching which are attributes of viruses and phytoplasma. xv
xvi
Symptoms are always the expression of some disorders of the plants which result from being invaded by the biotic factors like viruses, fungi, phytoplasma, nematodes etc and also the result from human manipulations (Safdar et al., 2010). In mango decline the first symptoms that appear in declining tree is leaf drooping that shows that the nutrients flow has stopped inside the vascular tissues. Actually the decline causing fungi enter through the wounded roots or stem and invade the vascular tissues. They keep growing and form the tylosis in the vascular bundles, thus blocking the flow of nutrients. Resultantly, the nutrients keep depositing in the air pockets which when overflow are ruptured the bark and oozing out as thick dark fluid. In most of the affected trees the stain of overflowing fluid were seen. When the stem of the affected trees are scraped after peeling of the bark, the dark brown to grey cankers are found
(Kazmi et al., 2005; Fateh et al., 2006; Masood et al., 2010). Similar symptoms have been observed in guava but the leaf drooping wass not as much apparent as in mango.
However, leaf drying and leaf drooping is found in many trees. The dark stains at the base of trees are also observed in guava (Rangaswami, 1984).
8.3. MYCOFLORA ISOLATED FROM THE DECLINE AFFECTED TREES
A number of fungi have been isolated from different decline affected trees and different plant parts. These include Botryodiplodia, Botryosphaeria, Nattrassia mangiferae,Fusarium, Alternaria, Pythium, Phytophthora and Ceratocystis species.
These fungi have been found infecting the trees singly or in combination. For exampleBotryodiplodia, Botryosphaeria and Nattrassia mangiferae were mostly found in the twigs and branches of all affected trees. They may be the cause of twig blight and branch die back in the affected trees. Nattrassia and Botryodiplodia were also xvi
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observed on the bark as well as on the inner side of the bark where bark splitting occurred. Therefore, they might have an additional role in bark splitting.
FusariumandCeratocystis have been found in the vascular tissues of the plants to stop the nutrients inflow and cause the wilting symptoms (Fateh et al., 2016). However, it is impotant to note thatCeratocystis was only found from the vascular tissues of mango but it was not isolated from citrus and guava (Mirzaeeet al., 2003; Miskita et al., 2005;
Fateh et al., 2006;Masood et al., 2010).
8.4. PATHOGENICITY OF THE IMPORTANT DECLINE CAUSING
PATHOGENS
Two different methods were adopted to check the pathogenicity of all the four commonly occuring fungi individually as well as in combination. These four fungi were Ceratocystis, Nattrassia, Botryodiplodia and Fusarium sp. Individual fungi except Ceratocystis was not able to produce mortality at all. However, the combinations of these fungi caused mortality in the green house potted plants.
Infact,Botryosphaeria species are endophytic in nature and become aggressive only when they are combined with the virulent fungi. The opportunistic fungi when entered the plants' system through wound, they start producing decline symptoms in the plants.
Among the two methods, the root injury method was more appropriate as the root injury frequently produced the symptoms. This can be related to the fact that when ploughing is done under the tree canopies, the roots are damaged and they provide opening to the fungi to enter and cause tree decline (Al-Adawi et al., 2006; Urbez-
Torroset al., 2008).
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8.5. FACTORS AFFECTING THE GROWTH AND DEVELOPMENT OF
NATTRASSIA SP.
In the samples taken from citrus plants, the growth ofNattrassia isolates was least affected by the temperature range from 20-28oC. However, in case of mango and guava, the temperature below 25 oC reduced the growth of the fungi (Alizada et al., 2000). This may be due to the fact thatN. mangiferae isolates have been adopted to high temperatures in the tropical areas as compared to subtropical or temperate regions. Mycelial growth was seen more in the isolates of Nattrassia when provided alternate light and dark periods as compared to continuous light or continuous dark periods. However, again the isolates from mango and guava did not show significant differences in any condition of light and dark (Elliott and Edmonds, 2003). Nattrassia isolates from citrus were the least affected with the pH range of 5.5 to 6.5. However, in mango and guava the isolates performed well at pH up to 6.00. The results are contradictory to the actual climatic conditions especially in the mango orchards where soil pH is more than 7 but N. mangiferae keeps growing. Among four culture media used for the growth of N. mangiferae, it only preffered to grow maximum on potato dextrose agar and malt extract agar media (El Atta and Aref, 2013). Other two media i.e. carrot juice agar and stem decoction agar media produced less growth of the fungus. However, it was found that these two media give pure growth of Ceratocystis which is also a decline causing fungi (Fateh and Kazmi, 2010).
8.6 MORPHOLOGICAL CHARACTERISTICS OF NATTRASSIA SP. FROM
CITRUS, MANGO AND GUAVA
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The similarity between isolates of mango and guava shows that there has been a shift of attack of N. mangiferae either from mango to guava or otherwise.
The chances are that these are the same fungal isolates which attack both trees as both are grown in tropical climate and as discussed earlier that sometimes guava is intercropped in mango orchards and hence there has been a transfer from mango to guava (Sutton and Dyko, 1989; El Atta and Nouri, 2014).
It has been found during the pathogenicity tests that the symptoms produced by
N. mangiferae in both trees were almost similar, which is again a confirmation of their common origin (Fateh et al., 2016). In case of citrus, the morphology of N. mangiferae was a little bit different. One of the reasons might be the difference in climate where citrus is grown and secondly, it can be due to the fact that in citrus the fungus has been reported to produce branch wilt with cracks in a particular check pattern, while in mango and guava they either cause branch dieback, branch canker or twig die back
(Mirzae et al., 2002; Saaiman, 1997).
However, the morphology of N. mangiferae isolates from all citrus, mango and guava had closed resemblance with madrone canker isolates in USA. Madrone isolates of N. mangiferae are characterized as hyaline, aseptate, guttulate conidia becoming 1- to 3-septate, verisicolored or brown. Conidia had thin-walls, mostly fusoid but occasionally oblong or clavate with truncate bases. The isolates differed in conidial length, width as well as in arthrospore size. Although there were statistically significant differences, there does not appear to be a geographical pattern in spore size or shape. The researchers in US conclude that N. mangiferae isolated from Pacific
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madrone belonged in the teleomorph genus Botryosphaeria and hence, diseases cause by this fungus can be managed using methods developed for other Botryosphaeria pathogens, such as B. dothidea(Elliott and Edmonds, 2003).
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SUMMARY
In Pakistan important fruit trees such as citrus, mango, guava etc are facing decline. Among these fruits, citrus and mango decline have been addressed strongly as they are important foreign exchange earnings and export items. Mango is being given attention in all aspects but citrus needs special attention. Due to poor quality citrus in
Pakistan we have restricted export markets. According to Pakistan Horticulture Export
Board, 20% of the citrus fruit is gone waste either due to diseases or post harvest mishandling (Humayun et al., 2005; Khanzada et al., 2004). Other fruit trees like
Guava and Loquat are also very famous fruit of Pakistan and are liked by all generations.
The common symptoms found in the orchards included the bark splitting gummosis, leaf drooping and discoloration, twig blight, die back and the eventual tree mortality.
Decline in different forest and fruit trees involve different pathogens e.g. fungi such as Ceratocystis fimbriata, Armillaria sp., Nattrassia magniferae, Lasiodiplodia theobromae, Phoma sp., Phomopsis sp., Fusarium sp., viruses such as Citrus Tristeza
Virus (CTV), nematodes especially in case of citrus decline (Tylenchulus semipenetrans) and in mango (Hemicriconemoides, Longidorus sp.), bacteria such as
Pseudomonas sp. and phytoplasma. Among these pathogens the fungus Nattrassia magniferae has been reported from almost all of the forest and fruit trees facing decline.
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Citrus decline incidence, severity and disease index were found to be variable in district Sargodha. Maximum mean disease incidence was recorded in tehsil
Sargodha (94.06%) followed by tehsil Shahpur (93.33%), similarly, mean disease severity was the maximum in tehsil Sargodha (1.47), Bhalwal (1.38). Maximum disease index was observed in tehsil Sargodha206 (29.41%) followed by tehsil Shahpur
(26.75%), Bhalwal (23.77%), Sahiwal (19.28%), and Kot Momin (15.80%). On the other hand minimum disease index was found in tehsil Sillanwali (10.36%).
In mango maximum mean disease severity 2.70 was observed in district
Bahawalpur followed by Khanewal (2.30). Similarly maximum mean disease incidence (100% ) was found in Khanewal followed by Bahawalpur (99.30%). Disease index was found maximum in Bahawalpur (53.30%) followed by Khanewal (46.50%).
Results show that maximum guava decline severity was recorded in district
Sheikhupura (0.90) followed by district Kasur (0.78). Minimum severity was observed in district Nankana Sahib (0.57). Maximum guava decline incidence was found in district Kasur (62.89%) followed by Sheikhpura (61.30%) while minimum incidence was observed in district Nankana Sahib (56.29%). However, regarding disease index district Kasur and Sheikhpura were not too far (18.00 and 17.40 respectively but minimum disease index (14% ) was recorded in district Nankana Sahib.
Mycofloral studies showed that in all kind of decline of citrus, mango and guava mostly common pathogens are involved in producing range of symptoms like, leaf drooping, bark splitting, twig or branch dieback, stem canker and eventual tree mortality. Moreover, an important fact found was that mostly N. mangifereae, L. theobromae and Botryosphaeria sp. were found in case of twig or branch dieback. xxii
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However, Fusarium sp. and Ceratocystis sp. were found in the vascular regions.
Two different methods were adopted to check the pathogenicity of
Ceratocystis, Nattrassia, Botryodiplodia and Fusarium sp. Individual fungi except
Ceratocystis was not able to produce mortality at all. However, the combinations of these fungi led the green house potted plant to mortality. In fact Botryosphaeria species are endophytic in nature and become aggressive only when they are combined with the virulent fungi.
During physiological studies it was found that Nattrassia isolates growth was least affected by the temperature range from 20-28oC. However, in case of mango and guava, the temperature below 25 oC reduced the growth of the fungi (Alizada et al.,
2000). This may be the reason that the Nattrassia isolates have been adopted to high temperature in the tropical areas as compared to subtropical or temperate regions.
Edmonds, 2003).Nattrassia isolates from citrus were least affected with the pH range of 5.5 to 6.5. However, in mango and guava the isolates have been preferring pH up to
6.00. The results are contradictory to the actual climatic conditions especially in the mango orchards where soil pH is more than 7. butNattrassia keeps growing. This may be due to low pH of fluent in the twigs and branches of mango tree which favour the growth of this fungus. Among four culture media used for the growth of Nattrassia, it only like to grow maximum on potato dextrose agar (PDA) and malt extract agar
(MEA) media (El Atta and Aref, 2013). Other two media i.e. carrot juice agar and stem decoction agar media produced less growth of the fungus. However, it was found that these two media give pure growth of Ceratocystis which is also a decline causing fungi
It can be concluded from all of the above discussion that Nattrassia mangiferae xxiii
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is a common pathogen of citrus mango and guava decline. It is mostly involved in the twig blight or die back of the trees. However, when it is combined with other aggressive isolates it can contribute towards the mortality of trees. There are few morphological differences among the isolates from mango and citrus, therefore it emphasizes to have a molecular study in future to see whether, it is Nattrassia mangiferae or other species of Nattrassia are also prevailing in Pakistan. Similarly there is different preference of light, temperature and pH for the development of the growth. The factors which favour the disease development other than fungi are intercropping in the orchards; ploughing under tree canopy causing root injury or damage by other field implements; application of fresh or partially decomposed farm yard manure, deficiency of the nutrients because of poor nutrition plans. Ignorance to pruning, which is a best tool to avoid pests and diseases and produce high quality fruits without compromising on yield. Above all the interst of the growers to manage their orchards.
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