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UNIVERSITI PUTRA MALAYSIA

PLANT DENSITY AND VARIETAL EFFECTS ON

DIAPORTHE/PHOMOPSIS DISEASES INFECTION OF

SOYBEAN AND ITS IMPACT ON SEED QUALITY

SAMIYEH RAEISI PARSAEI

FP 2011 42

PLANT DENSITY AND VARIETAL EFFECTS ON

DIAPORTHE/PHOMOPSIS DISEASES INFECTION OF

SOYBEAN AND ITS IMPACT ON SEED QUALITY


DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2011

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PLANT DENSITY AND VARIETAL EFFECTS ON DIAPORTHE/PHOMOPSIS

DISEASES INFECTION OF SOYBEAN AND ITS IMPACT ON SEED

QUALITY

By


Thesis Submitted to the School of Graduate Studies, University Putra Malsysia,

In fulfillment of the Requirements for the Degree of Doctor of Philosophy

May 2011

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DEDICATION

Dedicated to:

My dear and beloved family,

And

All workingman and toiler farmers

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfillment of

the requirement for the degree of Doctor of Philosophy

PLANT DENSITY AND VARIETAL EFFECTS ON DIAPORTHE/PHOMOPSIS

DISEASES INFECTION OF SOYBEAN AND ITS IMPACT ON SEED

QUALITY

By


May 2011

Chairman: Associate Professor Adam Puteh, PhD.

Faculty: Agriculture

Soybean is one of the most important oilseed crops with high protein for human and

animal consumptions. Environmental factors, field management and varieties affect on

the quality of harvested seed. Storage environment and its duration are also known to

influence pathogen survival in stored seed. A comprehensive study was undertaken at

University Putra Malaysia to evaluate the effect of plant density and seasonal variations

in relation to Diaporthe / Phomopsis complex (DPC) on seed yield components and

quality. Three soybean varieties namely AGS190, Deing and Pershing at 300,000 and

600,000 plants per hectare were grown in the field for two seasons in 2009. Seed

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germination and vigour were determined using standard germination (SG), tetrazolium

(TZ) and electrical conductivity (EC) tests. Seed yield components were determined at

harvest maturity growth stages (R8). Culture plate method and Scanning Electron

Microscopy (SEM) were used to evaluate the incidence of Phomopsis seed decay

(PSD) and the progression and colonization of DPC in different parts of soybean plant

from R3 until R8 plant growth stages. Ten soybean seed lots which were stored at 0°C

up to 13 years were used to evaluate the presence of DPC and to identify the isolates.

Morphological and molecular methods were used to determine the longevity and

frequency of DPC isolates on stored and freshly harvested soybean seeds.

Analysis of data showed significant differences for higher seed yield, 100 seed weight

and seed viability in two seasons and they were higher in first planting season. Planting

density affected on pod number per plant, height of plant, PSD infection and seed

viability and vigour significantly. Higher pod number was recorded at lower plant

density which was 30 pods per plant. The first node height increased at higher plant

density. PSD infection was more at higher plant density, so seed viability and vigour

were higher at lower plant density. The infection of PSD was 35% at higher plant

density, whereas it was 27.5% at lower plant density. The varieties showed significant

differences for all parameters. AGS190 with the highest 100 seed weight could produce

1839 kg seed yield per hectare. This variety also had the highest percentage of infection

to DPC (41%), so it showed the lowest seed viability and vigour. The highest pod

number was recorded for Deing variety, but it produced the lowest seed yield due to

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very small size seed. PSD infection was negatively correlated with standard

germination and tetrazolium test and showed a positive correlation with electrical

conductivity.

Scanning electron microscopy micrograph revealed that fungi colonization and

progression started at different growth stage in different parts of plant. The infection

started from R4 growth stage as fungi hypha whereas alpha conidia of fungi were

observed at R8 growth stage on the stem. Hypha was observed in the pod at growth

stage R5 and mycelium invaded inside the pod through the stomata at growth stage R6,

but alpha conidia invaded through the stomata at R7 growth stage. The seed was the

last part that was infected. The mycelium penetrated inside the seed through the hilum

and cracks on the surface of seed. Based on SEM observations, DPC infection can be

predicted sooner than R6 growth stage, so controlling DPC presence and infection

between R4-R5 can control the disease before damage being inflicted on the seed.

Seven isolates of DPC were detected, identified and characterized based on

morphological and molecular methods in soybean seeds for the first time in Malaysia.

The isolates which were identified molecularly were chosen from seeds that showed

>10% infection based on culture plate method. Most of the isolates identified, belonged

to Phomopsis longicolla, and all of them were registered in National Center for

Biotechnology Information (NCBI). The longevity of isolates in storage was found to

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be <9 years which suggested that Diaporthe /Phomosis sp. can survive up to 9 years in

cold storage( 0°C).

The results of this study showed that high quality soybean seed can be produced in wet

and warm tropical environments like Malaysia, but it should be planted at a proper time

in a production year and harvested on time, with no delay. Continuous rainfall during

late plant growth stage can cause more infection of PSD which can severely affect seed

quality and decrease the germinability of harvested seeds. Overall, 300,000 plant ha-1

is

an acceptable planting density with proper plant managements to produce high quality

soybean seeds in Malaysia.

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Abstrak tesis yang dikemukan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falasafah

TANAMAN KEPADATAN DAN PENGARUH KELAINAN PADA INFEKSI

PERYAKIT-PERYAKIT DIAPORTHE/PHOMOPSIS DARI KEDELAI DAN

DAMPAK PADA KUALITAS BIJI

Oleh


May 2011

Pengerusi: Profesor Madya Adam Puteh, PhD.

Fakulti: Pertanian

Soya merupakan salah satu tanaman biji minyak yang paling penting dengan protein

tinggi untuk dimakan manusia dan haiwan. Faktor persekitaran, pengurusan lapangan

dan kelainan berpengaruh terhadap high benih dituai. Simpanan persekitaran dan

durasinya juga diketahui mempengaruhi kelangsungan hidup patogen pada biji yang

disimpan. Sebuah kajian menyeluruh dilakukan di Universiti Putra Malaysia untuk

menilai pengaruh kerapatan tanaman dan variasi bermusim dalam kaitannya dengan

Diaporthe / kompleks Phomopsis (DPC) pada komponen hasil biji dan high. Tiga

kelainan soya iaitu AGS190, Deing dan Pershing di 300.000 dan 600.000 tanaman per

hektar ditanam di lapangan selama dua musim di 2009. perkecambahan benih dan

vigor ditentukan menggunakan perkecambahan standard (SG), tetrazolium (TZ) dan

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konduktiviti elektrik (EC) ujian. komponen hasil benih ditentukan pada tahap

pertumbuhan tuai jatuh tempo (R8). Budaya pinggan kaedah dan Mikroskop Elektron

(SEM) digunakan untuk menilai kejadian pembusukan benih Phomopsis (PSD) dan

perkembangan dan penjajahan DPC di berbagai bahagian tanaman soya dari R3 sampai

R8 tahap pertumbuhan tanaman. Sepuluh lot benih soya yang disimpan dalam 0 ° C

hingga 13 tahun digunakan untuk menilai kewujudan DPC dan untuk mengenalpasti

isolat. Morfologi dan kaedah molekul digunakan untuk menentukan umur panjang dan

kekerapan DPC isolat disimpan dan baru dituai biji soya.

Analisis data menunjukkan perbezaan yang signifikan bagi hasil biji lebih tinggi, berat

100 biji dan viabilitas benih dalam dua musim dan mereka lebih tinggi pada musim

tanam pertama. Penanaman kepadatan berpengaruh terhadap jumlah polong pada

tanaman, tinggi tanaman, jangkitan PSD dan viabilitas benih dan kekuatan secara

signifikan. jumlah polong lebih tinggi tercatat sebanyak kerapatan tanaman yang lebih

rendah yang 30 polong pada tanaman. Ketinggi an node pertama meningkat pada

kerapatan tanaman yang lebih tinggi. PSD jangkitan lebih pada kerapatan tanaman

yang lebih tinggi, sehingga viabilitas benih dan vigor lebih tinggi pada kerapatan

tanaman yang lebih rendah. Jangkitan PSD adalah 35% pada kerapatan tanaman yang

lebih tinggi, sementara itu 27,5% pada kerapatan tanaman yang lebih rendah. Varietas

menunjukkan perbezaan yang nyata untuk semua parameter. AGS190 dengan berat 100

biji tertinggi dapat menghasilkan 1.839 kg hasil biji per hektar. Kelainan ini juga

mempunyai peratusan tertinggi jangkitan untuk DPC (41%), sehingga menunjukkan

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viabilitas benih dan vigor terendah. Jumlah polong tertinggi tercatat untuk Deing, tapi

ini menghasilkan benih terendah kerana saiz biji sangat kecil. PSD jangkitan

berkorelasi negatif dengan standard dan uji perkecambahan tetrazolium dan

menunjukkan korelasi positif dengan konduktiviti elektrik.

Scanning elektron mikrograf mikroskop mendedahkan bahawa cendawan penjajahan

dan pembangunan bermula pada tahap pertumbuhan yang berbeza di pelbagai bahagian

tanaman. Jangkitan bermula dari tahap pertumbuhan R4 sebagai hifa cendawan

sedangkan alpha konidia cendawan diamati pada fasa R8 pertumbuhan pada batang.

Hifa diamati di R5 polong pada tahap pertumbuhan dan miselium menyerang di dalam

polong melalui stomata pada pertumbuhan tahap R6, tapi konidia alpha menyerbu

melalui stomata pada R7 tahap pertumbuhan. Benih adalah bahagian terakhir yang

dijangkiti. Miselium menembus di dalam benih melalui hilus dan retak pada

permukaan benih. Berdasarkan pengamatan SEM, DPC jangkitan boleh diramal lebih

cepat dari R6 tahap pertumbuhan, sehingga kewujudan DPC dan pengendalian

jangkitan antara R4-R5 dapat mengawal penyakit sebelum kerosakan yang ditimbulkan

pada benih.

Tujuh isolat DPC dikesan, dikenalpasti dan ditandakan berdasarkan kaedah morfologi

dan molekul dalam biji kedelai untuk pertama kalinya di Malaysia. Isolat yang

dikenalpasti molekul dipilih daripada biji yang menunjukkan jangkitan >10%

berdasarkan kaedah plate budaya. Sebahagian besar isolat dikenalpasti, milik longicolla

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Phomopsis, dan semua dari mereka yang berdaftar di Pusat Nasional untuk

Biotechnology Information (NCBI). Umur panjang isolat dalam simpanan dijumpai <9

tahun yang menunjukkan bahawa Diaporthe / sp Phomosis. boleh bertahan hingga 9

tahun dalam cold storage (0°C).

Keputusan kajian menunjukkan bahawa biji soya high tinggi boleh dihasilkan dalam

lingkungan tropika basah dan hangat seperti Malaysia, tetapi harus ditanam pada masa

yang tepat dalam satu tahun pengeluaran dan tuai tepat waktu, dengan tidak ada

penangguhan. curah hujan terus menerus selama tahap pertumbuhan tanaman akhir

boleh menyebabkan jangkitan lebih dari PSD yang sangat boleh menjejaskan kualiti

benih dan menurunkan perkecambahan biji dituai. Secara keseluruhan, tanaman

300.000 ha-1 adalah kerapatan tanam diterima dengan pengurusan kilang yang tepat

untuk menghasilkan benih soya yang berkualiti tinggi di Malaysia.

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ACKNOWLEDGEMENTS

At first my praises and endless thanks be to God, who gave me the strength and

opportunity to complete my study. I hope and promise to use my knowledge for the

people who need it. I would like to convey my deepest appreciation to my supervisor,

Associate Professor Dr. Adam Puteh, for his supervision, valuable advices and helpful

suggestions in my study, especially his skilful and kind guidance for my thesis writing

that resulted in completion of my thesis. Thanks and sincere appreciation goes to my

committee members, Associate Professor Dr Kamaruzaman Sijam and Dr Nur Ashikin

Psyquay Abdullah, for their supports, help and kind permission to work in their

laboratories and use all apparatus and facilities.

I would like to express my honest thanks to the staff members of Microbiology and

Physiology laboratories, for their kind helps and also providing facilities and material

for my research, especially, Mrs., Junaina and Mr. Davud. I am certainly grateful to

Mr. Zol from seed technology laboratory for his assistance and all facilities that he

supplied for my field and laboratory experiments, and also greatly thanks to Mr.

Rahman for his valuable assistance in preparing and managing the field.

I know my duty, sincerely thank from all people in my dear country, Iran, for their

support and create this opportunity for me to continue my supplementary study,

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including, Agriculture Research Organization, Seed and Plant Improvement Institute,

and especially Agricultural Research Center of Golestan.

My heartiest thanks and love to my beloved family for their constant emotional and

physical supports, patients and endless helps and encouragements. Moreover, I deeply

thank anybody who has helped me with continuing my study

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I certify that a Thesis Examination Committee has met on 6 May 2011 to conduct the

final examination of Samiyeh Raeisi Parsaei on her thesis entitled “Plant Density and

Varietal Effects on Diaporthe/Phomopsis Diseases Infection of Soybean and its Impact

on Seed Quality” in accordance with Universities and University College Act 1971 and

the constitution of the Universiti Putra Malaysia [P. U. (A) 106] 15 March 1998. The

committee recommends that the student be awarded the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Abdul Shukor b Juraimi PhD

Associate Professor

Faculty of Agriculture

University Putra Malaysia

(Chairman)

Zainal Abdin b Mior Ahmad PhD

Associate Professor



(Internal Examiner)

Mohd Ridzwan b Abd Halim PhD

Associate Professor



(Internal Examiner)

Moshir Rahman PhD

Associate Professor

Department of agronomy

Bangladesh Agriculture University

(External Examiner)

-------------------------------

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 27 June 2011

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The

members of Supervisor Committee were as follows:

Adam Puteh, PhD

Associate Professor



(Chairman)

Kamaruzaman Sijam, PhD

Associate Professor



(Member)

Nur Ashikin Psyquay Abdullah, PhD

Senior Lecturer



(Member)

-----------------------------------------------

HASANAH MOHD GHAZALI. PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: July 2011

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DECLARATION

I declare that the thesis is my original work except for questions and citations, which

have been duly acknowledged. I also declare that it has not been previously and it not

currently submitted for any other degree at Universiti Putra Malaysia or any other

institutions.

________________________


Date: 6 May 2011

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS x

APPROVAL xii

DECLARATION xiv

LIST OF TABLES xix

LIST OF FIGURES xx

LIST OF ABBREVIATIONS xxii

CHAPTER

I INTRODUCTION

1

II LITERATURE REVIEW

2.1 Soybean growth and development 5

2.1.1 Seed dry weight 5

2.1.2 Seed moisture 5

2.2. Seed yield and quality 6

2.2.1 Seed yield components 6

2.2.2 Viability and vigour 8

2.3 Factors affecting seed quality 9

2.3.1 Genetics 9

2.3.2 Management and environments during production 11

2.3.3 Soybean seed borne pathogens 14

2.4 Seed quality tests 16

2.4.1Viability and vigour tests 16

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2.4.2 Seed – borne pathogen tests 18

2.5 Dynamic of Diaporthe/Phomopsis infection during seed

Production

21

2.6 Pathogen survival during seed storage 24

III SEED YIELD AND QUALITY OF SOYBEAN IN RELATION

TO PHOMOPSIS SEED DECAY

3.1 Introduction

26

3.2 Materials and Methods 29

3.2.1 Seed yield components and seed yield 31

3.2.2 Seed quality evaluation 31

3.2.3 Standard germination test 32

3.2.4 Tetrazolium chloride test 32

3.2.5 Electrical conductivity test 32

3.2.6 Bioassay for Phomopsis Seed Decay 33

3.3 Data Analysis 34

3.4 Results 34

3.4.1 The effects of planting density on seed yield components 36

3.4.2 The effect of plant density on seed quality 39

3.4.3 Relation between Phomopsis incidence and seed viability 42

3.5 Discussion 44.

IV THE DYNAMIC OF Diaporthe/Phomopsis COMPLEX

INFECTION DURING REPRODUCTIVE STAGES

4.1 Introduction

48

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4.2.1 Culture plate method 52

4.2.2 Scanning Electron Microscopy (SEM) 52

4.3 Results 53

4.3.1 The progression of DPC infection based on culture plate method 53

4.3.2 Movement of DPC in the plant 55

4.4 Discussions 62

V LONGEVITY AND ISOLATES OF Diaporthe/Phomopsis SPECIES

IN STORED SOYBEAN SEEDS

5.1 Introduction

66


5.2.1 Infection of Diaporthe/phomopsis complex in seed lots by

culture plate method

69

5.2.2 Identification of DPC species on infected soybean seeds 69

5.2.3 Identification of DPC isolates based on morphological

characteristics

70

5.2.4 Identification of DPC isolates in soybean seeds using

molecular method

70

5.3 Results 72

5.3.1 DPC seed infection of stored seeds 72

5.3.2 Diaporthe/Phomopsis species based on morphological identification

of stored seed

73

5.3.3 Diaporthe/Phomopsis isolates based on morphological identification

of stored seed

75

5.3.4 Occurrence and persistence of Diaporthe/Phomopsis isolates of

stored seed

77

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5.3.5 Diaporthe/Phomopsis isolates based on molecular identification

of stored seed

78

5.4 Discussions 82

VI GENERAL DISCUSSION, CONCLUSION AND

RECOMMENDATION

85

REFERENCES 91

APPENDIX 108

BIODATA OF STUDENTS 117

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