pathogenic fungi (Umashankar et al., 2012; Tchameni et al., 2011; Coudert et al.,. 2010; Joshi et al., 2010). It is likely that improvement in root growth was the ...
EFFICACY OF Trichoderma spp. CONSORTIUM AS POTENTIAL BIOCONTROL AGENTS OF GANODERMA DISEASE OF OIL PALM, AND INVOLVEMENT OF ANTIOXIDANT ENZYMES AND TOTAL PHENOLIC CONTENT
By
HABU MUSA
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirements for the Degree of Master of Science
January 2017
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DEDICATION
The work in this thesis is dedicated to the memory of loving daughter and son, to my entire family members and my beloved wife, Rashida Umar for their boundless love, understanding, encouragement and patience throughout my study.
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master of Science
EFFICACY OF Trichoderma spp. CONSORTIUM AS POTENTIAL BIOCONTROL AGENTS OF GANODERMA DISEASE OF OIL PALM, AND INVOLVEMENT OF ANTIOXIDANT ENZYMES AND TOTAL PHENOLIC CONTENT
By
HABU MUSA
January 2017
Chairman Faculty
: Nusaibah binti Syd Ali, PhD : Agriculture
Oil palm (Elaeis guineensis) is an economically important cash crop, grown primarily for its oil and has become one of the main oil crop in the world. Unfortunately, a major constrain faced by oil palm industry particularly in Malaysia and Indonesia is the devastating disease known as Ganoderma disease caused by Ganoderma spp. To date, this disease has been causing considerable oil palm yield losses and collapse of oil palm trees, consequently affecting its contribution to the producer’s economy. Thus, research on sustainable and eco-friendly remedy to counter Ganoderma disease is on the upsurge to avoid the current control measures via synthetic fungicides. Trichoderma spp. have been the most studied and valued microbes as biological control agents in an effort to combat a wide range of plant diseases sustainably. In addition, knowledge on Trichoderma’s mechanism employed to act as biological control agent, could be useful in improving on their ability as potent biological control agents against Ganoderma spp. Therefore, in this current study, the potential of Trichoderma spp.(Trichoderma asperellum, Trichoderma harzianum, and Trichoderma virens) as a consortium approach was evaluated as biological control agents against Ganoderma disease on oil palm with these objectives: i) To characterize and identify the selected Trichoderma spp. via morphological and molecular characterization, ii) To evaluate their bio-inoculant potential as oil palm seedling treatment for the control of Ganoderma disease and iii) To study the effects on vegetative growth and suppression of Ganoderma boninense infection in oil palm seedlings treated with Trichoderma spp. as single or consortium application.
i
Prior to in vitro assessment, all Trichoderma isolates studied were subjected to morphological and molecular identification. Antagonistic effects of Trichoderma spp. against G. boninense growth evaluated via dual culture test and culture filtrate test. The results demonstrated that the percentage inhibition of radial growth (PIRG) of G. boninense was of >75% in both in-vitro assays. In addition, ability of Trichoderma spp. in producing fungal cell wall degrading enzymes such as chitinase, glucanase, β-1, 3-glucanases and protease were also assessed. All the isolates studied demonstrated potential for the production of cell wall degrading enzymes.
However, to measure the potential in Ganoderma disease suppression and enhancement of vegetative growth, an in-vivo trial of six months duration was conducted on oil palm seedlings via G. boninense artificial inoculation treated with single species and consortium of Trichoderma spp. in a nursery condition. The consortium of Trichoderma spp. applied found to be the most effective treatment in suppressing Ganoderma disease with 83.03% and 89.16% from the foliar and bole symptoms respectively. Besides that, the consortium application of Trichoderma spp. exhibited tremendous enhancement in the oil palm seedling vegetative growth parameters such as plant height, stem girth, leaf area, frond count, shoot and root (fresh and dried weight) with significant difference at P 75% dalam kedua-dua ujian in vitro tersebut. Di samping itu, keupayaan Trichoderma spp. dalam menghasilkan enzim yang mampu mendegradasi dinding sel kulat seperti enzim chitinase, glucanase, β-1, 3-glucanases dan protease juga telah dinilai. Berdasarkan keputusan kajian yang diperolehi, kesemua isolat yang dikaji telah mempamerkan potensi dalam menghasilkan enzimenzim tersebut.
Walaubagaimanapun, untuk mengukur potensi dalam merencatkan jangkitan penyakit Ganoderma serta peningkatan dalam pertumbuhan vegetatif, satu kajian secara in-vivo selama enam bulan telah dijalankan ke atas anak sawit melalui kaedah inokulasi G. boninense secara artifisial dan dirawat dengan spesies tunggal dan gabungan Trichoderma spp. dalam keadaan nurseri yang terkawal. Rawatan Trichoderma spp. yang diaplikasi secara gabungan telah dikenalpasti sebagai rawatan yang paling efektif dalam merencatkan jangkitan Ganoderma dengan memberikan 83.03% dan 89.16% melalui simptom-simptom pada daun dan akar padat masing-masing. Selain itu, aplikasi Trichoderma spp. secara gabungan juga telah mempamerkan peningkatan yang memberangsangkan pada parameterparameter pertumbuhan vegetatif anak sawit seperti ketinggian pokok, ukur lilit batang, keluasan daun, bilangan daun, pucuk dan akar (berat segar dan berat kering) dengan perbezaan signifikan pada P TCCGTAGGTGAACCTGCGGAAGGATCATTATCGAGTTTTGACTGGGTTGTAGCTGGCCTT CCGAGGCATCGTGCACGCCCTGCTCATCCACTCTACACCTGTGCACTTACTGTGGGTTAT AGATCGTGTGGAGCGAGCTCGTTCGTTTGACAAGTTCGCGAAGCGCGTCTGTGCCTGCGT TTTATCACAAACACTATAAAGTATTAGAATGTGTATTGCGATGTAACGCATCTATATACA ACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAA GTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTT GGTATTCCGAGGAGCATGCCTGTTTGAGTGTCATGAAATCTTCAACCTACAATCTCTTTG CGGTTTTTGTAGGCTTGGACTTGGAGGCTTGTCGGTCTTTTATTGATCGGCTCCTCTCAA ATGCATTAGCTTGGTTCCTTTGCGAATCGGCTGTCGGTGTGATAATGTCTACGCCGCGAC CGTGACGCGTTTGGCGAGCTTCTAACCGTCCCGTTATTGGGACAACTCTTATGACCTCTG ACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
Sequence of Trichoderma asperellum TCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCAATGTGA ACGTTACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACC AGGCGCCCGCCGGAGGAACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTATTTCT TACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTC TGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCG AGCGTCATTTCAACCCTCGAACCCCTCCGGGGGATCGGCGTTGGGGATCGGGACCCCTCA CACGGGTGCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGT TTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGA AATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA
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Sequence of Trichoderma brevicompatum TCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCCAATGTG AACGTTACCAAACTGTTGCCTCGGCGGGATTTCTGCCCCGGGCGCGTCGCAGCCCCGGAC CAAGGCGCCCGCCGGAGGACCAATTTACAAACTCTTTTGTATATCCCATCGCGGATTCTT TACATTCTGAGCTTTCTCGGCGCTCCTAGCGAGCGTTTCGAAAATGAATCAAAACTTTCA ACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGT GAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTC TGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCGT TGGGGATCGGCACTTACCTGCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTC CTGCGCAGTAGTTTGCACACTCGCACCGGGAGCGCGGCGCGTCCACGGCCGTAAAACAAC CCAAACTTCTGAAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATAT CAATAAGCGGAGGA
Sequence of Trichoderma harzianum TCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCAATGTGA ACGTTACCAAACTGTTGCCTCGGCGGGATCTCTGCCCCGGGTGCGTCGCAGCCCCGGACC AAGGCGCCCGCCGGAGGACCAACCAAAACTCTTATTGTATACCCCCTCGCGGGTTTTTTT TTATAATCTGAGCCTTCTCGGCGCCTCTCGTAGGCGTTTCGAAAATGAATCAAAACTTTC AACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATG TGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATT CTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGGTCGGCG TTGGGGATCGGCCCTCCCTTAGCGGGTGGCCGTCTCCGAAATACAGTGGCGGTCTCGCCG CAGCCTCTCCTGCGCAGTAGTTTGCACACTCGCATCGGGAGCGCGGCGCGTCCACAGCCG TTAAACACCCAACTTCTGAAATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTA AGCATATCAATAAGCGGAGGA
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Sequence of Trichoderma harzianum TCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTTTACAACTCCCAAACCCAATGTGA ACGTTACCAAACTGTTGCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAGCCCCGGAACC AGGCGCCCGCCGGAGGAACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGACGTATTTCT TACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTC TGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGA ATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCG AGCGTCATTTCAACCCTCGAACCCCTCCGGGGGATCGGCGTTGGGGATCGGGACCCCTCA CACGGGTGCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGT TTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGA AATGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAG GA
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Appendix C: Cellulase, protease and chitinase activities a
b
a) Trichoderma asperellum and Trichoderma brevicompatum Cellulose activities. a
b
a) Trichoderma asperellum and Trichoderma brevicompatum protease activities.
a
b
a) Trichoderma asperellum and Trichoderma brevicompatum chitinase activities
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Appendix D1: Trichoderma spp. colony growth (cm) on different fungus growing media Isolates PDA CMA Czapek agar MEA T. harzianum 8.17b 7.43a 5.52ab 7.80a T. asperellum 8.57a 6.80ab 6.03a 7.90a T. virens 7.10c 7.70a 5.16b 7.52a T. brevicompatum 6.50d 5.60b 4.31c 6.11b Means represented with the same letter in a column are not significantly different by Duncan Multiple Range Test.
Appendix D2: Effect of Temperature (°C) on Trichoderma spp. colony growth (cm) on PDA medium Isolates 15°C 20 °C 25°C 30°C 35°C T.harzianum 2.1b 3.92a 5.09b 5.35b 2.96a T.asperellum 2.51a 3.30b 5.86a 6.07a 2.82a T.brevicompactum 2.20b 2.84c 5.18b 5.48b 2.51b T. virens 2.61a 3.86a 5.13b 5.42b 2.45b Means represented with the same letter in a column are not significantly different by Duncan Multiple Range Test.
Appendix D3: Effect of pH on Trichoderma spp. colony growth (cm) on PDA medium 4 Isolates 6 7 8 9 T.harzianum 3.71a 6.20b 7.77a 6.17ab 4.39c T.asperellum 3.25b 6.73a 7.87a 6.35a 6.03a T.brevicompactum 3.81a 6.03b 7.01b 5.70b 4.39b T. virens 3.93a 6.13b 7.57a 6.04ab 6.50a Means represented with the same letter in a column are not significantly different by Duncan Multiple Range Test.
Appendix D4: Hydrolytic enzymes activities of Trichoderma spp. Isolates Cellulose Chitinase Protease glucanase T. harzianum 45.15b 51.70b 43.57b 46.57bc T. asperellum 55.50a 53.52a 51.03a 51.17a T. virens 55.22a 49.78b 43.85b 49.99ab T.brevicompactum 35.74c 47.16c 40.97b 45.84c Means represented with the same letter in a column are not significantly different by Duncan Multiple Range Test.
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Appendix D5: Effect of Trichoderma spp. on plant height 1
Treatment 2 1 3 2 4 3 5 4 6 5 7 6 35.47ab 41.73ab 47.96b 57.63b 65.10ab 68.25ab T1 35.99ab 41.09ab 48.80b 55.23b 62.23bc 65.62bc T2 35.75ab 41.79ab 48.84b 53.34b 59.06b 62.85c T3 38.35a 44.52a 53.37a 62.72a 66.57a 69.86a T4 33.11b 38.38b 43.07c 47.14c 47.81c 51.53d T5 27.06c 33.50c 38.02d 40.70d 42.85d 43.53e T6 Means represented with the same letter in a column are not significantly different by Tukey's Studentized Range (HSD) Test. [T1= Treated with T. asperellum + G. boninense + plant; T2= Treated with T. harzianum + G. boninense + plant; T3= Treated with T. virens + G. boninense + plant; T4= Treated with consortium of T1 + T2+ T3 + G. boninense + plant; T5= Negative control + plant; T6= Positive control (only G. boninense) + plant] Appendix D6: Effect of Trichoderma spp. on Stem girth (mm) 9 Treatment 10 1 11 2 3 4 5 6 10.82a 11.50a 14.12a 15.19a 17.10a 17.65a T1 9.77ab 12.10ab 13.98a 14.96a 16.19ab 16.92a T2 8.99ab 11.54ab 13.71a 14.61a 15.43b 16.44a T3 9.98a 13.00a 14.41a 15.46a 17.25a 17.72a T4 9.77bc 10.71bc 11.19b 12.66b 13.61c 16.43b T5 9.05c 9.96c 10.23b 10.42c 10.95d 11.77c T6 Means represented with the same letter in a column are not significantly different by Tukey's Studentized Range (HSD) Test. [T1= Treated with T. asperellum + G. boninense + plant; T2= Treated with T. harzianum + G. boninense + plant; T3= Treated with T. virens + G. boninense + plant; T4= Treated with consortium of T1 + T2+ T3 + G. boninense + plant; T5= Negative control + plant; T6= Positive control (only G. boninense) + plan
815 13 12 14
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Appendix D7: Effect of Trichoderma spp. on oil palm seedling leave area index 16 17 21 22 19 18 20 Treatment 1 2 3 4 5 6 109.52a 138.34cd 235.60a 254.83bc 269.91b 288.30bc T1 113.62a 168.90b 237.22a 261.20ab 276.26ab 303.17bc T2 110.45a 162.51c 241.02a 271.58ab 286.40ab 312.77ab T3 121.26a 214.39a 240.69a 322.71a 337.45a 361.48a T4 100.60a 149.10cd 182.20b 219.81bc 232.84bc 253.17d T5 77.57a 134.47d 171.52b 192.59bc 206.45c 229.11d T6 Means represented with the same letter in a column are not significantly different by Tukey's Studentized Range (HSD) Test. [T1= Treated with T. asperellum + G. boninense + plant; T2= Treated with T. harzianum + G. boninense + plant; T3= Treated with T. virens + G. boninense + plant; T4= Treated with consortium of T1 + T2+ T3 + G. boninense + plant; T5= Negative control + plant; T6= Positive control (only G. boninense) + plant]. Appendix D8: Effect of Trichoderma spp. on oil palm seedlings front count unit 23 24 29 28 25 27 26Treatment 1 2 3 4 5 6 3.33a 4.00a 5.00a 7.00ab 7.67bc 8.00bc T1 3.33a 4.00a 4.67ab 6.33ab 7.00ab 7.33bc T2 3.33a 4.00a 4.33ab 6.33ab 7.00ab 8.00ab T3 3.00a 4.00a 5.00a 7.67a 8.00a 8.67a T4 2.67a 3.67a 4.33ab 6.00b 6.33cd 6.67cd T5 2.33a 3.00b 3.67c 5.67b 6.00c 6.00d T6 Means represented with the same letter in a column are not significantly different by Tukey's Studentized Range (HSD) Test. [T1= Treated with T. asperellum + G. boninense + plant; T2= Treated with T. harzianum + G. boninense + plant; T3= Treated with T. virens + G. boninense + plant; T4= Treated with consortium of T1 + T2+ T3 + G. boninense + plant; T5= Negative control + plant; T6= Positive control (only G. boninense) + plant]
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Appendix E1: Summary of ANOVA table for the effect of different media on the growth of Trichoderma spp. Sources PDA trt Re CMA trt Rep CZAPEK trt Rep MEA trt rep
Mean Square
F Value
Pr > F
R-Square value
2.71444444 0.54166670
5.01
0.0001
0.949475
2.62333333 0.50833333
5.16
0.0283
0.659313
1.58555556 0.09416667
16.84
0.0008
0.863279
2.34000000 0.886364 0.0004 0.11250000 trt treatment; rep replication.
0.886364
Appendix E2: ANOVA table for the effects of temperature on Trichoderma spp. colony growth on potato dextrose agar (PDA) Sources t-15 trt rep t-20 trt rep t-25 trt rep t-30 trt rep t-35 trt rep
Mean Square
F Value
Pr > F
R-Square value
0.08840333 0.00711389
12.43
0.0040
0.911939
0.47238167 0.03559722
13.27
0.0034
0.917071
0.24970833 0.01841389
13.56
0.0032
0.918704
0.20647000 0.01844167
11.20
0.0053
0.903193
0.10922833 10.48 0.0063 0.897239 0.01042500 t temperature, trt treatment and rep replication
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Appendex E3: ANOVA table for the effects of pH on Trichoderma spp. colony on potato dextrose agar (PDA) Sources
Mean Square
P4 trt 0.26510000 rep 0.08002500 P6 trt 0.29467500 rep 0.04493333 P7 trt 0.44645556 rep 0.06786667 P8 trt 0.22800833 rep 0.07703333 P9 trt 2.47158889 rep 0.08562500 trt treatment, rep replication, P pH,
F Value
Pr > F
RSquare value
3.31
0.0780
0.554023
6.56
0.0151
0.710922
6.58
0.0149
0.711559
2.96
0.0977
0.526055
28.87
0.0001
0.915430
Appendex E3: ANOVA table for In vitro antagonistic Screening Trichoderma spp. against G. boninense Dual culture Source
DF Sum of Squares Mean Square F Value Pr > F
trt
3
918.609079
306.203026
rep
20
1071.036917
53.551846
Total 23 R-Square
1989.645996 0.461695
5.72 0.0054
Appendex E5: ANOVA tables for Activity profile of hydrolytic enzymes secreted by the antagonists Trichoderma spp. Chitinase Source
DF Sum of Squares Mean Square F Value Pr > F
trt
3
977.490375
325.830125
rep
16
284.423480
17.776467
Total R-Square
19
1261.913855 0.774609 93
18.33 F
trt
3
72.0426550
24.0142183
1.01
0.4148
rep
16
381.1464400
23.8216525
Total R-Square
19
453.1890950 0.158968
Protease Source
DF
Sum of Squares
Mean Square
F Value
Pr > F
trt
3
279.4831400
93.1610467
4.77
0.0146
rep
16
312.2655600
19.5165975
Total R-Square
19
591.7487000 0.472300
DF
Sum of Squares
trt
3
100.6678950
33.5559650
rep
16
129.9958000
8.1247375
Total R-Square
19
230.6636950 0.436427
Glucanase Source
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Mean Square F Value
Pr > F
4.13 0.0240
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BIODATA OF STUDENT
Habu Musa was on 23rd February, 1984 in Maiduguri, the capital of Borno state, in the Northeast Nigeria. He received his primary education in Maimusari primary school, Maiduguri and secondary education in Government secondary school Kukawa, Borno state. In 2000, he was first matriculated in Kashim Ibrahim college of Education, Maiduguri and received Nigeria certificate in education (NCE) in 2003. He continued his bachelor in University of Maiduguri and graduated with Bachelor of Agriculture (B. Agriculture) with Honours in Crop Protection in 2009. He worked on Root knot nematodes (Meloidogyne spp) extraction, identification and alternate control measures in the Northeast, Nigeria. Soon after, graduation he was enlisted for National Youth Service and served in Enugu state ministry of Agriculture as agricultural officer.
He joined the service of the Federal University Dutse, Jigawa state, Nigeria in 2013 as Graduate assistant in the department of Crop Production and was awarded TETFund Fellowship for Oversea Study in 2014. He registered for Master of Science in Plant Pathology in University Putra Malaysia under the supervision of Dr Nusaibah Binti Syd Ali, department of Plant Protection.
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10 LIST OF PUBLICATIONS Assessment on Trichoderma spp. mixture as a potential biocontrol agent of Ganoderma boninense infected oil palm seedlings. Submitted to Journal of Oil Palm Research. JOPR-2016-0108.R3.
Topical overview on the mechanism of Trichoderma spp. as biological control agent of plant diseases. Submitted to Journal of Tropical Agricultural Science. (JTAS-1003-2016.R1)
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