UNIVERSITI PUTRA MALAYSIA ASSOCIATION OF RED-TIP OF PINEAPPLE LEAVES WITH NUTRIENT DEFICIENCY VIJIANDRAN A/L JUVA RAJAH FP

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1 UNIVERSITI PUTRA MALAYSIA ASSOCIATION OF RED-TIP OF PINEAPPLE LEAVES WITH NUTRIENT DEFICIENCY VIJIANDRAN A/L JUVA RAJAH FP

2 ASSOCIATION OF RED-TIP OF PINEAPPLE LEAVES WITH NUTRIENT DEFICIENCY By VIJIANDRAN A/L JUVA RAJAH Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science August 2007

3 xix LIST OF FIGURES Figure Page 1 The Red-tip Of Pineapple Leaves 9 2 Stages Of Red-tip Development on Pineapple Leaves 9 3 Nitrogen deficient plant at 145 DAP 34 4 Plant exhibiting severe nitrogen deficiency at 270 DAP 35 5 Plants exhibiting phosphorus deficiency at the initial stages at 150 DAP 36 6 Severe phosphorus deficiency exhibited at 270 DAP 37 7 A close up view of an older leaf deficient in phosphorus 37 8 Early symptoms of potassium deficiency, noted after 2 nd fertilisation 38 9 Severe deficiency of potassium at 270 DAP General view of a plant deficient in potassium at 270 DAP Plants deficient in calcium, after 2 nd fertilisation Plant deficient in calcium, after 4 th fertilisation A close look at magnesium deficient plants at 145 DAP Plants deficient in sulphur after 2 nd fertilisation Close look at the red tip formed on sulphur deficient leaves at 150 DAP 16 Plants treated without the input of sulphur at 270 DAP with some red-tip formations red-arrows) after the completion of the 4 th fertilisation round Total number of leaves of plants for various treatments compared at each period of sampling 50

4 xx 18 General Plant Height of various treatments compared at each period of sampling Total number of leaves for T1 and T2 at various stages of sampling Mean leaf dry weight of T1 and T2 at various stages of sampling Stem circumference of T1 and T2 at various stages of sampling Mean Stem Dry Weight of T1 and T2 at various stages of sampling 23 Red-tip percentage in the leaves of T1 and T2 at various stages of sampling Nitrogen uptake in D-leaf at different stages of plant growth Phosphorus uptake in D-leaf in different stages of plant growth Potassium uptake in D-leaf in different stages of plant growth Calcium uptake in D-leaf in different stages of plant growth Magnesium uptake in D-leaf in different stages of plant growth Sulphur uptake in D-leaf in different stages of plant growth Zinc uptake in D-leaf in different stages of plant growth Copper uptake in D-leaf in different stages of plant growth Manganese uptake in D-leaf in different stages of plant growth Nitrogen uptake in stem at different stages of plant growth Phosphorus uptake in stem at different stages of plant growth Potassium uptake in stem at different stages of plant growth Calcium uptake in stem at different stages of plant growth Magnesium uptake in stem at different stages of plant growth 83

5 xxi 38 Sulphur uptake in stem at different stages of plant growth Zinc uptake in stem at different stages of plant growth Copper uptake in stem at different stages of plant growth Manganese uptake in stem at different stages of plant growth Sampling areas of Pineapple leaf samples for microscopic work Confocal Microscope view of the green area of pineapple leaves 44 Confocal Microscope view of the red-tip area of the pineapple leaf. 45 Phloem region of green area magnified at 1000 X using the Confocal Microscope 46 Phloem region of red-tip area magnified by 1000 X using the Confocal Microscope 47 Sieve element cells of the phloem region of the green area under the TEM at the magnification of X 48 Sieve element cells of the phloem region of the red-tip area under the TEM at the magnification of X 49 Magnification of X of the strange bodies found in the sieve element cells in the red-tip area of the pineapple leaves. 50 Mycoplasma like organisms found in citrus likubin. Globular structures marked with the yellow arrow 51 Mycoplasma like organisms of sweet potato witches broom. Note the numerous bodies located in the sieve elements

6 ii DEDICATION This thesis is dedicated to :- Both my parents Mr. and Mrs. Juva Rajah-Punithavathy the souls responsible for what I am today My wife Christina Vijiandran who had shared all the wonderful and troubled moments of my life since the very first day we met To the contributors and potential contributors of any kind of knowledge that is beneficial to mankind and is as well in harmony with nature

7 iii Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science ASSOCIATION OF RED-TIP OF PINEAPPLE LEAVES WITH NUTRIENT DEFICIENCY By VIJIANDRAN A/L JUVA RAJAH August 2007 Chairman: Associate Professor Ahmad Husni Mohd. Hanif, PhD, Faculty: Agriculture The red-tip of pineapple leaves which affected the Gandul variety was sighted in the early 1990 s in Peninsula Plantations, Simpang Renggam, Johore where it has been found that about 10% or more of its leaves starting from the tip had turned red. It was speculated that it was due to sulphur deficiency as a result of the change of nitrogen carriers from ammonium sulphate to urea. This study was carried out with three main objectives: (i) to investigate whether the red-tip problem is related to nutrient deficiency through Missing Element Experimental Technique and thus identify the nutrients that are involved, (ii) to investigate the effect of ammonium sulphate and urea as the nitrogen carriers on the redtip of pineapple leaves and also the plant performance in terms of vegetative growth and fruit yield at field conditions and (iii) to examine and compare the cell structural

8 iv differences between the normal (green) and infected (red-tip) part of the pineapple leaf from cv. Gandul The Missing Element experiment was carried out in the glasshouse to achieve the first objective while the second objective was achieved through a field experiment in Simpang Renggam, Johore. Further to the above experiments, the red-tip area and the green area of the leaves were also viewed under a Confocal and Transmission Electron Microscope to look for any differences between them at the cellular level which covered the third objective of the study. Visual observations of the plants from the first experiment could not reproduce the redtip as found in Simpang Renggam for all treatments including plants treated without the input of sulphur though there was some occurrence of the red-tip in these plants at 150 days after planting (DAP). The deficiency symptom of other eliminated nutrients was mostly similar to that reported in literature and there were no similarities seen compared with the red-tip phenomenon. Vegetative growth variables studied from the destructive sampling carried out at 180 and 300 days after planting showed significant differences between treatments only at the later stage of plant growth, the second stage of sampling. The first experiment indicated that sulphur deficiency was not the primary cause for the occurrence of the red-tip phenomenon in the pineapple leaves. Plants grown in the field from both treatments, i.e. plants fertilised with ammonium sulphate as nitrogen fertiliser (Treatment 1, T1) and plants fertilised with urea as nitrogen

9 v fertiliser (Treatment 2, T2) did not show any significant differences between them in terms of vegetative growth, plant nutrient uptake, soil nutrient concentrations and yield. There were also no significant differences between T1 and T2 in the red-tip percentage at all sampling periods except one but further soil and plant nutrient analysis did not show any significant differences. Besides that, the uptake trend of the macro and micro nutrients was also not significant for both treatments and the levels had indicated healthy growth for both treatments. The sulphur and other nutrient concentrations for plant and soil were well above the critical point for deficiency to occur and the continuous occurrence of red-tip in plants treated with input of ammonium sulphate gave a doubt whether sulphur is the main cause of the phenomenon. The insignificant yield between the two treatments had further increased the doubt. The second experiment had further strengthened that the deficiency of sulphur is not the cause of the red-tip in the pineapple leaves. With the above findings, observations carried out using the Confocal Microscope of both the green and red-tip area of the pineapple leaves revealed the presence of some blockages in the phloem cells of the red-tip area which was not found in the green area. Further magnification of this area using the Transmission Electron Microscope revealed presence of some globular structures in the sieve element cells of the red-tip area similar to the infection of Mycoplasma Like Organisms (MLO). The presence of these structures had most likely blocked the movement of nutrients through sieve element cells thus causing the disintegration of chlorophyll to anthocyanin causing the red-tip of pineapple leaves. However further trials are needed to confirm this speculation.

10 vi The study had given an understanding that the red-tip phenomenon is not caused by the deficiency of sulphur or any other plant nutrients but due to the presence of the globular structures in leave cells which may be MLO.

11 vii Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains ASSOCIATION OF RED-TIP OF PINEAPPLE LEAVES WITH NUTRIENT DEFICIENCY Oleh VIJIANDRAN A/L JUVA RAJAH Ogos 2007 Pengerusi : Profesor Madya Ahmad Husni Mohd Hanif, PhD Fakulti : Pertanian Fenomena hujung merah daun nanas yang telah menjangkiti varieti Gandul telah ditemui pada awal 1990an di Peninsula Plantations, Simpang Renggam, Johor dimana 10% atau lebih daripada daunya telah berubah menjadi merah bermula dari hujungnya. Spekulasi yang dijangka adalah kekurangan sulfur disebabkan oleh perubahan pembawa nitrogen dari ammonium sulfat kepada urea. Kajian ini telah dijalankan dengan tiga objektif utama iaitu (i) untuk mengkaji sama ada masalah hujung merah daun berkaitan dengan kekurangan nutrien melalui Teknik Eksperimen Pelupusan Nutrien dan seterusnya mengenalpasti nutrien yang mempengaruhinya, (ii) untuk mengkaji kesan ammonium sulfat and urea sebagai pembawa nitrogen pada hujung merah daun nanas serta prestasi tumbuhan dari segi pertumbuhan vegetatif serta hasil dalam keadaan lapangan dan (iii)

12 viii untuk mengkaji serta membandingkan perubahan struktur sel diantara bahagian biasa (hijau) dan yang dijangkiti (hujung merah) daun nanas cv. Gandul. Eksperimen untuk Pelupusan Nutrien telah dijalankan di Rumah Kaca, untuk memenuhi objektif pertama, manakala objektif kedua kajian ini dicapai melalui satu ekperimen di lapangan di Simpang Renggam, Johor. Tambahan kepada kedua-dua eksperimen tersebut, bahagian hujung merah serta bahagian hijau daun nanas telah diperhatikan di bawah Mikroskop Confocal dan Transmisi Elektron untuk melihat perbezaan diantara keduadua bahagian tersebut diperingkat selular untuk memenuhi objektif ketiga. Pemerhatian visual pokok-pokok yang ditanam untuk eksperimen pertama, tidak boleh menghasilkan hujung merah seperti yang diperhatikan di Simpang Renggam terutamanya pokok-pokok yang dirawat tanpa input sulfur meskipun terdapat sedikit pembentukan hujung merah pada pokok-pokok tersebut iaitu 150 hari selepas penanaman. Simptom kekurangan bagi nutrien lain yang diperhatikan telah menunjukan pemerhatian yang sama seperti dalam literatur dan tidak mempunyai persamaan dengan fenomena hujung merah. Parameter-parameter pertumbuhan vegatatif yang dikaji daripada persampelan pemusnahan yang dijalankan pada 180 dan 300 hari selepas penanaman hanya menunjukan perbezaan rawatan yang bererti pada peringkat lewat pertumbuhan tanaman, iaitu pada peringkat persampelan yang kedua. Eksperimen pertama menunjukkan kekurangan sulfur bukan punca utama yang membawa kepada fenomena hujung merah didalam daun nanas.

13 ix Pokok-pokok yang ditanam dilapangan daripada kedua-dua rawatan iaitu pokok-pokok yang dibaja dengan ammonium sulfat sebagai baja nitrogen (Rawatan 1, T1) dan pokokpokok yang dibaja dengan urea sebagai baja nitrogen (Rawatan 2, T2) tidak menunjukkan sebarang perbezaan bererti diantara mereka dari segi pemerhatian pertumbuhan vegetatif, pengambilan nutrien oleh pokok, kepekatan nutrien di dalam tanah serta hasil pengeluaran. Tiada sebarang perbezaan bererti diantara rawatan T1 dan T2 dalam peratus hujung merah yang pada semua tempoh persamplen kecuali satu dimana pemerhatian lanjutan dengan analisis tanah dan nutrien tanaman yang tidak menunjukkan perbezaan yang beerti. Selain itu, tren pengambilan nutrient makro dan mikro juga tidak menunjukkan sebarang perbezaan yang bererti diantara kedua-dua rawatan serta menunjukkan pertumbuhan pokok yang sihat. Tahap kepekatan sulfur dan nutrien lain didalam pokok serta tanah telah melebihi tahap kritikal untuk membolehkan masalah kekurangan nutrien berlaku serta kewujudan hujung merah berterusan pada pokok-pokok yang diberi input ammonium sulfat telah mewujudkan suatu kesangsian sama ada sulfur adalah penyebab utama fenomena tersebut. Hasil pengeluaran yang tidak bererti bagi kedua-dua rawatan tersebut turut menambah kesangsian ini. Eksperimen kedua telah memperkuatkan pendapat bahawa kekurangan sulfur bukan merupakan punca kejadian fenomena hujung merah pada daun nanas. Pemerhatian dengan Mikroskop Confocal untuk kedua-dua bahagian hujung hijau dan merah daun nanas, telah menunjukkan kehadiran sedikit sumbatan di sel-sel floem pada bahagian hujung merah daun dimana sumbatan sebegini tidak wujud dibahagian hijau daun. Pembesaran lanjut bahagian ini dengan menggunakan Mikroskop Transmisi

14 x Elektron pula menunjukkan kehadiran sedikit struktur berbentuk globular di sel-sel elemen penapis pada bahagian hujung merah yang mempunyai persamaan seperti jangkitan Organisma Seperti Mikoplasma (MLO). Kewujudan struktur sebegini diramalkan telah menghalang perjalanan nutrien melalui sel elemen penapis yang seterusnya membawa kepada perubahan klorofil kepada antosianin yang seterusnya menyebabkan hujung merah daun nanas. Walaubagaimanapun kajian yang lebih lanjut diperlukan untuk mengesahkan spekulasi ini. Kajian ini telah memberi suatu pemahaman bahawa fenomena hujung merah tidak dipengaruhi oleh kekurangan sulfur atau nutrien-nutrien pokok lain tetapi disebabkan oleh kewujudan struktur globular didalam sel-sel daun yang berkemungkinan jangkitan MLO.

15 xi ACKNOWLEDGEMENTS First of all I would like to thank the Almighty for the inner motivation and strength poured into me throughout the duration of this study. My deepest gratitude also goes to my beloved parents, Mr and Mrs Juva Rajah and my loving wife Christina for boosting my confidence and providing me the push to complete this thesis. I also would like to thank my siblings Navindran and Thaneswary for all the support during the tough times encountered while completing this thesis. To all my family members, thank you for your constant support given to me in one way or another especially to cousins George Wong and Kalyani for the trouble taken to edit this thesis at the initial stages and my nephews Sudesh and Previnash for recreating the smile in my face when the going gets tough. My sincere appreciation goes to Assoc. Prof. Dr. Ahmad Husni who had given me all the guidance, advice, help and support needed all through the duration of the study besides having the confidence in me. The contributions on Assoc. Prof Dr. Aminuddin Husin and Assoc. Prof. Dr. Mahmud Tengku Muda on their various field of expertise is also deeply acknowledged. The help rendered by Puan Sarimah, En. Junaidi, Madam Suleka, Mr. Ho and other staffs of both the Department of Land Management especially Soil Fertility Lab 2 and the Microscopy Unit, Institute of Bioscience is never forgotten. A big thank you to Dr. Osumanu Haruna Ahmed for all his help, guidance and brotherly advice given to me all these years I had known him. I would also like to appreciate the management of Peninsula Plantations Sendirian Berhad especially to Mr. Koh Tsu Koon for allowing us

16 xii to carry out our experiment in the estate and also for the supply of planting materials and media needed for the study. Their kind hospitality given to us during all our visits to the estate is also deeply appreciated. I would also like to take this opportunity to thank my employers, United Plantations Berhad for the employment during the needy times of which it had gave me the financial strength to complete my studies. Last but not least to each and every individual who helped me in one way or another in completing this study.

17 xiii APPROVAL I certify that an Examination Committee has met on 28 August 2007 to conduct the final examination of Vijiandran a/l Juva Rajah on his Master of Science thesis entitled "Association of Red-tip of Pineapple Leaves with Nutrient Deficiency" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations The Committee recommends that the student be awarded the relevant degree. Members of the Examination Committee are as follows: Mohd Khanif Yusop, PhD Professor Department of Land Management Faculty of Agriculture Universiti Putra Malaysia (Chairman) Samsuri Abdul Wahid, PhD Lecturer Department of Land Management Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Ganesan Vadamalai, PhD Lecturer Department of Plant Protection Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Mohammad Selamat Madom, PhD Unit Head Planting Material, Seed and Livestock Breed Production Unit Malaysian Agricultural Research and Development Institute (External Examiner) HASANAH MOHD GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

18 xiv DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions. VIJIANDRAN JUVA RAJAH Date: 12 December 2007

19 xv TABLE OF CONTENTS Page DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ii iii vii xi xiii xiv xv xviii xix CHAPTER I INTRODUCTION 1 II REVIEW OF LITERATURE 3 Background of the Pineapple Industry 3 Pineapple Cultivation in Malaysia 4 The Pineapple Plant 4 The Pineapple Leaves 6 Red-tip of Pineapple Leaves 8 Essential Plant Nutrient Elements and It s Effect on the Growth of Pineapples 10 Nitrogen 11 Phosphorus 13 Potassium 14 Calcium 16 Magnesium 17 Sulphur 18 Peat Soils 19 Physical Characteristics of Peat 20 Chemical Characteristics of Peat 22 Fertility of Peat Soils 23 Mycoplasma Like Organisms 26 III INVESTIGATION OF THE RED TIP OF PINEAPPLE LEAVES THROUGH MISSING ELEMENT EXPERMENTAL TECHNIQUE 28 Introduction 28 Objective 28 Materials and Methods 29 Location of Experimental Site and Planting Materials Used 29 Experimental Designs and Treatments 30 Fertilisation Scheme 31 Sampling and Data Recording 32

20 xvi Results and Discussion 33 Visible Nutrient Deficiency Symptoms 33 First Destructive Sampling 45 Second Destructive Sampling 46 Vegetative Growth Comparison 48 Conclusion 54 IV V COMPARISON ON THE EFFECTS OF AMMONIUM SULPHATE AND UREA ON THE RED TIP OF PINEAPPLE LEAVES IN FIELD CONDITIONS 55 Introduction 55 Objective 55 Materials and Methods 56 Experimental Location 56 Fertilisation 56 Sampling 57 Variables Recorded 58 Plant Samples Preparation 58 Soil Samples Preparation 59 Plant Parts and Ash Analysis 59 Soil Chemical Analysis 61 Statistical Analysis 62 Results and Discussion 62 Total Leaves 62 Leaf Dry Weight 64 Stem Circumference 65 Stem Dry Weight 66 Red-tip Percentage 68 Plant Nutrient Concentrations and Uptake 69 Soil Nutrient Concentrations 86 Yield 90 Conclusion 91 OBSERVATION OF THE RED-TIP OF PINEAPLLE LEAVES UNDER CONFOCAL AND TRANSMISSION ELECTRON MICROSCOPE 93 Introduction 93 Objective 93 Materials and Methods 94 Sample Preparation 94 Results and Discussion 98 Confocal Microscope Observations 98 Transmission Electron Microscope Observations 101 Conclusion 106

21 xvii VI SUMMARY AND CONCLUSIONS 107 BIBLIOGRAPHY 109 BIODATA OF THE AUTHOR 114

22 xviii LIST OF TABLES Table 1 Range and average percentage of important elements in organic soils. 2 Nutrient content of peat used as planting medium for the Missing Element Experiment Page Treatments for Missing Element Experimental Technique 31 4 Effect of treatments on total leaves production, leaf dry weight, stem dry weight and stem circumference at first destructive sampling 5 Effect of treatments on total leaves production, leaf dry weight, stem dry weight and stem circumference at second destructive sampling Fertilisation programme for T1 and T Mean Nutrient Content in D-leaves at various Stages of Sampling for T1 and T2 8 Mean Nutrient Content in Stem at various Stages of Sampling for T1 and T2 9 Nutrient concentrations in soil samples at 0 25 cm depth at various stages of sampling for T1 and T

23 CHAPTER I INTRODUCTION Pineapples (Ananas comosus), originated from South America (Othman and Subhadrabanthu, 1995; Barthlomew and Malézieux, 1994), like many other crops in Malaysia, have been domesticated, improved and developed since pre-historic times, often with no records of agronomy (Othman and Subhadrabanthu, 1995). Originated from the Bromiliaceae family, the cultivation of pineapples in Malaysia is rather unique as it is cultivated largely on peat (Ahmed et al., 2002). In Malaysia, the crop is planted in an area of 6400 hectares (2003) for both Table consumption and canning. Seventy eight percent of the total area is under pineapple estates while the balance belongs to small holders. Over the last few years, the production of canned pineapples in Malaysia has decreased. Malaysia was producing 189,700 tonnes of fresh fruit in 1991 at its peak and dropped to 73,500 tonnes (forecast) in Most of the pineapples were produced by the estates which accounted between 77 to 92 percent of total country production from 1990 to The decrease in the production is mainly due to the reduction of planting areas especially among the small holders where the area decreased by about 30% over the same period mentioned above (AGRIQUEST 2006). Pineapple planted in Malaysia serves two main purposes, Table consumption and canning. The Sarawak pineapple, which originated from the Cayenne variety and Morris from the Queen variety are commonly planted for Table consumption while the Gandul and Masmerah from the Spanish variety for canning purposes (Selamat and Ramlah,

24 2 1993). The Gandul, which is largely planted in the Johore area, has been selected for its good yielding even though there are several shortcomings (Selamat and Abdul,1996). Over the years, numerous researches have been carried on the pineapple fruit and the plant itself with the aim of improving the yield thus increasing the income of farmers and making the industry profitable. In addition to that, various studies have been also been carried out to control diseases and pests infecting the plants such as mealy bug wilt (Dysmicoccus brevipes) the common pineapple pest. Recently, a new problem had arisen which has affected the gandul variety called red-tip of pineapple leaves. The red-tip phenomenon was first sighted in Peninsula Plantations and speculations were that it was caused by nutrient deficiency. It is believed that this phenomenon had caused a reduction in yields which is an effect of nutrient deficiency which led to this study. This study was aimed to look into the red-tip of pineapple leaves in the nutrient point if view with three main objectives, (i) to investigate whether the Red-tip problem is related to nutrient deficiency through Missing Element Experimental Technique, (ii) to investigate the effect of both ammonium sulphate and urea as the nitrogen carriers on the red-tip of pineapple leaves and also plant performance in terms of vegetative growth and fruit yield under field conditions and (iii) To examine and compare the cell structural differences between the normal (green) and infected (red-tip) part of the pineapple leaf from cv. Gandul.

25 3 CHAPTER II REVIEW OF LITERATURE Background of The Pineapple Industry Pineapple is one of the 45 genera and 2000 species available in the Bromeliaceae family (Nakasone and Paull, 1998). Originated from South America and has been well developed and now its world trade is dominated by the canned fruit. The world pineapple production fluctuated between to million standard cases (4,516 pineapples per standard case) from 1990 to Thailand and Philippines continued to lead the world in total canned pineapple export, accounting for 44.6% and 14.5% respectively (AGRIQUEST, 2006). Pineapple has been the oldest agricultural export sector in Malaysia but unfortunately it never really took off and had always been a subsidiary crop for the country. The export of this product by Malaysia however decreased significantly from 6% in 1990 ( standard cases) to 2.6% in 2003 which accounts for standard cases (AGRIQUEST, 2006). The area under pineapple crop in Malaysia is decreasing steadily in recent years as the growers shift to more profitable and less intensive crops. But the decrease is all among the smallholdings because the nature of the industry makes the estate area more resistant to change, as most estate owners are also owners of the canning factories. Another reason for the drop in the area is clearly seen in the price of pineapple.