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Antimicrobial Properties of Methanol Crude Extract of Kacangma against Crop Pathogenic Fungi and Preliminary Report of Fungal Disease for Kacangma YONG LEE KIEN (39425) Bachelor of Science with Honours (Plant Resource Science and Management) 2015 Antimicrobial Properties of Methanol Crude Extract of Kacangma against Crop Pathogenic Fungi and Preliminary Report of Fungal Disease for Kacangma Yong Lee Kien 39425 This final project report is submitted in fulfilment of the requirement for the Degree of Bachelor of Science with Honours (Plant Resource Science and Management) Plant Resource Science and Management Department of Plant Science and Environmental Ecology Faculty of Resource Science and Technology University Malaysia Sarawak 2015 DECLARATION I hereby declare that the thesis is based on my original work. All the quotations and citations have been duly acknowledge. No portion of the work referred to this dissertation has been previously or concurrently submitted for any other degree programs in UNIMAS or other institutions of higher learning. _____________________ Yong Lee Kien Plant Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak (UNIMAS) APPROVAL SHEET Name of Candidate: Yong Lee Kien Title of Dissertation: Antimicrobial Properties of Methanol Crude Extract of Kacangma against Crop Pathogenic Fungi and Preliminary Report of Fungal Disease for Kacangma __________________________ Dr. Freddy Yeo Kuok San Supervisor __________________________ Dr. Mohamad Hasnul b. Bolhassan Co- supervisor __________________________ Dr Rebicca Edward Programme Coordinator Plant Resource Science and Management Faculty of Resource Science and Technology Universiti Malaysia Sarawak (UNIMAS) Acknowledgement First of all, I must express my greatest gratitude to thank and appreciate my supervisor, Dr. Freddy Yeo Kuok San, for his unceasing countenance, guidance, patience and motivation throughout my study. I am deeply grateful to him for providing helpful suggestion and comments on the draft documents leading to this thesis. His guidance will be remembered forever by me. Besides that, I extend my gratitude to my co-supervisor, Dr. Mohamad Hasnul b. Bolhassan, for his practical guidance and support during my entire study period. I would like to thank the Agriculture Research Centre Semenggok Sarawak for providing materials and experimental guidance for my study. Moreover, I would like to thank the Muara Tuang private farmer owner and other enthusiastic person in Batu Kawa residential area for providing kacangma planting areas to me. Also, my appreciation goes to the Department of Plant Science and Environmental Ecology for providing necessary equipment and facilities in my study. Last but most importantly, I dedicated this thesis to my beloved family. I will not forget their sincere love, moral support, countless prayers and endless efforts for my entire journey in university. This piece of work is also dedicated to my course mates, who had accompanied me for thousands of hours in my life. They had taught me to be strong and keep my faith, whenever I have met any difficulties. I Table of Contents Title Page I Acknowledgement II Table of Contents V List of Figures List of Tables V Abstract/ Abstrak VI Chapter 1: Research Background 1 1.1 Introduction 1.2 Problem Statements 3 1.3 Objectives 4 Chapter 2: Literature Review 2.1 Description of Kacangma 5 2.2 Phytochemical Extraction 6 2.3 Antimicrobial Plant Constituents 8 2.3.1 2.3.2 Chemical Properties of Leonurus sibiricus Chemical Properties of Leonurus japonicus 2.4 Crop Pathogenic Fungi 9 10 11 2.4.1 Glomerella cingulate 11 2.4.2 Fusarium oxysporum 11 2.4.3 Fusarium solani 12 2.4.4 Pyricularia oryzae 12 II 2.4.5 Pestalotiopsis sp. 13 2.4.6 Colletotrichum sp. 13 2.5 Disease of Kacangma 14 Chapter 3: Methodology 3.1 Plant Materials 16 3.2 Methanol Extraction 17 3.3 Dilution of Methanol Crude Extracts 18 3.4 Preparation of Crop Pathogenic Fungi 3.4.1 Potato Dextrose Agar 18 3.4.2 Oatmeal Agar 19 3.5 Inhibition Test on Selected Crop Pathogenic Fungi 20 3.6 Characterizing The Leaf Flavonoid Profile of Kacangma 21 3.7 Disease Description and Isolation of Causal Agent 3.7.1 Fungal Isolation 23 3.7.2 Morphological Identification of Isolated Fungi 23 3.7.3 Physiological Test of Temperature on Isolated Fungi 24 3.8 Pathogenicity Test of Kacangma Leaves 24 Chapter 4: Result 4.1 Crude Extract from Leonurus sibiricus and Leonurus japonicus Leaves 26 4.2 Antimicrobial Activity of Crude Extract Obtained from Leonurus sibiricus and Leonurus japonicus 26 III 4.3 Flavonoid Analysis of Kacangma 28 4.4 Preliminary Report of Kacangma Disease (Leonurus japonicus) 4.4.1 Symptoms of Disease 30 4.4.2 Morphological Characteristics of Fungi Isolated from The Disease of Leonurus japonicus 31 4.4.3 Temperature Effects on the Growth of Fungi Isolated from Leonurus japonicus 33 4.4.4 Pathogenicity Test on Leonurus sibiricus and Leonurus japonicus 34 Chapter 5: Discussion 5.1 Antimicrobial Testing of Crude Extract of Leonurus sibiricus and Leonurus japonicus 36 5.2 Flavonoid Analysis of Leonurus sibiricus and Leonurus japonicus 38 5.3 Preliminary Report of Kacangma Disease 40 Chapter 6 Conclusion and Recommendation 6.1 General Conclusion 41 6.2 Recommendation 42 References 43 Appendix 47 IV List of Figures Page 1.1 Title Leonurus sibiricus produce pink flower (a) and L. japonicus produce white flower (b). 2.1 Basic structure of flavonoid, C6C3C6 skeleton with two benzene rings (A and B) and one heterocyclic pyrane ring (C) (Cushnie & Lamb, 2005). 7 3.1 Experimental design of inhibition testing on each selected crop pathogenic fungi. 20 3.2 Experimental design of two dimensional paper chromatography. 22 3.3 Experimental design of pathogenicity test. 25 4.1 Leaf spot disease of the leaf of Leonurus japonicus 30 4.2 The morphology of isolated Fungus A, Fungus B and Fungus C from diseased leaves of Leonurus japonicus. 32 4.3 Pathogenicity test of Leonurus sibiricus and L. japonicus by Fungus A, Fungus B and Fungus C. 35 No. 1 List of Tables Title Structures of some selected antimicrobial flavonoids (Cushnie & Lamb, 2005). Symptoms and fruiting bodies structure of some common pathogenic fungi (Agrios, 2005). Page 7 4.1 Inhibition test reaction of Leonurus sibiricus against select crop pathogenic fungi. 27 4.2 Inhibition test reaction of Leonurus japonicus against select crop pathogenic fungi. 27 4.3 Overall comparison of flavonoid compounds of Leonurus sibiricus and L. japonicus collected from different locations and their tentative identification. 29 4.4 Kruskal-wallis tests on growth rate of Fungus A, Fungus B and Fungus C incubated in different temperature. 33 No. 2.1 2.2 V 15 Antimicrobial Properties of Methanol Crude Extract of Kacangma against Crop Pathogenic Fungi and Preliminary Report of Disease on Fungi Yong Lee Kien Plant Resource Science and Management Faculty Resource Science and Technology University of Malaysia Sarawak ABSTRACT Kacangma (Leonurus sibiricus and Leonurus japonicus) is a local medicinal plant in Sarawak, Malaysia. Antimicrobial properties of methanol crude extract of kacangma were examined against six crop pathogenic fungi namely, Fusarium oxysporum, Fusarium solani, Glomerella cingulata, Pyricularia oryza, Colletotrichum sp. and Pestalotiopsis sp.. No significant antimicrobial effects were observed among these tested crop pathogenic fungi. Flavonoid biosynthesis in kacangma plants that planted in three different locations (Batu Kawa residential area, Muara Tuang private farm and UNIMAS greenhouse) was analysed by Two-dimensional paper chromatography and compared. The leaf flavonoid profiles showed distinct polymorphism to one another which indicated the flavonoid biosynthesis in kacangma was environmentally dependent. Meanwhile, preliminary report on fungal disease of kacangma was studied. Leaf spot disease was infected in L. japonicus plant and the suspected causal agent was the unknown fungal species (Fungus B) which was positive in the pathogenicity test. In short, significant antimicrobial effects of kacangma against the crop pathogenic fungi were not showed, the flavonoid biosynthesis in kacangma was environment dependent and the suspected causal agent of leaf spot disease in L. japonicus was Fungus B. Keywords: Leonurus sibiricus, Leonurus japonicus, polymorphism, leaf spot disease ABSTRAK Kacangma (Leonurus sibiricus dan Leonurus japonicus) adalah ubat tumbuhan di Sarawak, Malaysia. Ciri-ciri antimikrob ekstrak metanol kasar kacangma telah diperiksa terhadap enam tanaman kulat pathogen iaitu, Fusarium oxysporum, Fusarium solani, Glomerella cingulata, Pyricularia oryza, Colletotrichum sp. dan Pestalotiopsis sp.. Tiada kesan antimikrob yang penting diperhatikan di kalangan tanaman diuji kulat pathogen. Biosintesis flavonoid dalam tumbuh-tumbuhan kacangma yang ditanam di tiga lokasi yang berbeza (Batu Kawa kawasan perumahan, Muara Tuang ladang swasta dan UNIMAS rumah hijau) dianalisis dengan kromatografi kertas dua dimensi dan dibandingkan. Profil flavonoid daun menunjukkan polymorfism berbeza antara satu sama lain yang menunjukkan biosintesis flavonoid dalam kacangma bergantung pada faktor-faktor alam sekitar. Sementara itu, laporan mengenai penyakit kacangma telah dikaji. Daun penyakit tempat telah dijangkiti oleh L. japonicus dan agen penyebab penyakit yang disyaki merupakan spesies kulat yang tidak diketahui (Kulat B) yang positif dalam ujian patogenisitasnya. Konklusinya, kesan antimikrob dari kacangma pada kulat patogen kurang berkesan, biosintesis flavonoid dalam kacangma adalah bergantung pada persekitaran dan agen penyebab penyakit daun L. japonicus disyaki adalah Kulat B. Kata Kunci: Leonurus sibiricus, Leonurus japonicus, polymorfism, daun penyakit tempat VI Chapter 1 Research Background 1.1 Introduction Kacangma is a well-known local medicinal plant in Sarawak, Malaysia. The family of kacangma is Lamiaceae which consist of many herbs that contain phytochemical compounds involved in various biological activities especially antimicrobial activities (Huang, Liao, & Tang, 2005; Topcu & Goren, 2007). The vernacular name, ‘kacangma’ is widely used in Malaysia which refer to two different species, namely Leonurus sibiricus and Leonurus japonicus. These herbaceous plants are native to the tropical Asia, India, China, Africa, Myammar and America (Mominul Islam & Kato-Noguchi, 2014). It can spread easily through seeds on a variety of soil types. The flowering of kacangma indicates the mature stage which normally achieved three months after sowing. L. sibiricus has pink flower whereas L. japonicus has white flower. (b) (a) Figure 1.1 Leonurus sibiricus produce pink flower (a) and L. japonicus produce white flower (b). 1 Numerous research studies on the chemical properties of kacangma had been carried on especially in the health science sector. Kacangma has antimicrobial, anti-platelet aggregation, anticancer and anti-inflammatory properties (Liang et al., 2013a). For the antimicrobial properties, the aqueous and ethanol extracts of L.sibiricus were able to inhibit the gram positive bacteria (Staphylococcus aureus), gram negative bacteria (Saccharomyces cerevisae) and pathogenic fungi (Aspergillus niger) (Chua & Aminah, 2011). These tested microorganisms are pathogens that cause skin disorder diseases on human. In reality, fewer researches were performed on the antimicrobial properties of kacangma against crop pathogenic fungi as compared to the human pathogenic fungi. This study focuses on testing the antimicrobial properties of kacangma against crop pathogenic fungi. The phytochemical compounds responsible for the antimicrobial properties of kacangma against pathogenic crop fungi may be potentially developed into biofungicide. Biofungicide is a form of biochemical pesticides that can be applied onto a specific disease whereby results a better disease prevention and management (Francis & Keinath, 2010). Biofungicide is environmental friendly which protects and maintains the equilibrium of ecosystem. One of the groups of phytochemical compounds which involved in antimicrobial activities is flavonoids. Flavonoids are produced in all vascular plants but their biosynthesis is influenced by diverse environmental factors (Downey, Dokoozlian & Krstic, 2014). The different environmental factors are referring to the variations of temperature, moisture, air movement and soil type. Thus, the leaf flavonoid contents of kacangma collected from three different locations were tested in this study and compared. 2 Kacangma is traditionally used by the Chinese as herbs that help in heart disorders, diuretics and dispel edema (Liang et al., 2013b; Mominul Islam & Kato-Noguchi, 2014). Moreover, it can also use to regulate menstrual disturbances and promote blood circulation (Liang et al., 2013b). Generally, it will be chopped, dried and cooked with ginger chicken soup. Nowadays, well packed dried kacangma are available at the local market and usually is made from L. sibiricus which is believes to be better than L. japonicus because of its pink flower. 1.2 Problem Statements Excessive practices of fungicides in plantation had triggered the eco-warning alarm and thereby the intervention of biofungicide was strongly promoted. The productions of potential phytochemicals for biofungicide may be are environment dependent including flavonoids. Kacangma is a medicinal plant that contains various phytohormones with potential antimicrobial properties. There are numerous research had carried on the antimicrobial properties of kacangma but limited on fungal diseases for kacangma. 3 1.3 Objectives The first aim was to test if the crude extract of kacangma (Leonurus sibiricus and Leonurus japonicus) will inhibit the growth of selected crop pathogenic fungi. The first objective was to obtain crude extracts of kacangma through methanol extraction. The second objective was to observe the inhibition effects of different concentration of kacangma crude extract against the growth of selected crop pathogenic fungi. The second aim was to test if any polymorphism of the flavonoid content will formed in kacangma that grew in different environment. The third objective was to compare and study the flavonoid profile of kacangma leaves collected from different locations. The third aim was to conduct preliminary report for fungal disease of kacangma. The fourth objective was to observe and describe the infection symptoms on diseased leaf of L. japonicus. The fifth objective was to isolate any associated fungal species from the diseased leaf of L. japonicus and conduct physiological test of different temperature. The sixth objective was to conduct pathogenicity test on healthy leaves of kacangma by the isolated fungal species. 4 Chapter 2 Literature Review 2.1 Description of Kacangma Kacangma is referred to L. sibiricus and L. japonicus in this study. Both species are belonging to the family of Lamiaceae. The two species are near identical morphologically except for their flower color. L. sibiricus has pink flower whereas L. japonicus has white flower. Both species are annual plants with square stem that is erect, stout and grooved (Datta, 1945). They grow upright to height ranges of 20 cm to 25 cm and the simple leaves are arranged in opposite dessucate pattern. This plant has polymorphic leaves which refer to ovate shaped leaves with crenate margin and cross-venulate venation and lobed shaped leaves with entire margin and palmate venation. Moreover, both of the plants develop beautiful spike pattern of inflorescence. Each flower is shaped in galeate with an oblong upper lip and one stigma that could yield four seeds (Edley, n.d.). L. sibiricus and L. japonicus can multiply through seed and cutting. The dark brown oval seeds can germinate in moist soils with adequate nutrients and sunlight within a week. Normally, the plant matures in approximately three months after sowing. 5 2.2 Phytochemical Extraction Extraction is a process to extract the potential biological components from the desired plants. Various extractions methods were designed and conducted depending on the type of plant samples and desired target compounds. Maceration and Soxhlet methods will extract phenolic compounds under high temperature condition in a long period, thus reduce phenolic compound contents in the extract (Dai & Mumper, 2010). These conventional extraction methods are not suitable used to obtain phenolic compounds which are easily hydrolysed and oxidised. Solvent extraction is a common extraction method. Polar solvent can be used to obtain polar components whereas non-polar solvents can be used to obtain non-polar components. The polar solvents, methanol was effectively used in extracting low molecular weight polyphenol compounds including the flavonoids with C6C3C6 skeleton (Figure 2.1) (Dai & Mumper, 2010; Waterman & Mole, 1994). Various different groups of flavonoids are formed by oxygenation and substitution on the C6C3C6 skeleton (Table 2.1). These compounds have three important properties namely antiviral, antifungal and antibacterial (Cushnie & Lamb, 2005). 6 Figure 2.1 Basic structure of flavonoid, C6C3C6 skeleton with two benzene rings (A and B) and one heterocyclic pyrane ring (C) (Cushnie & Lamb, 2005). Table 2.1 Structures of some selected antimicrobial flavonoids (Cushnie & Lamb, 2005). Flavonoid compounds Apigenin (Flavones) 6,8Diprenylgenistein (Isoflavones) Kaempherol Licochalcone A (Chalcones) Naringenin (Flavanones) Functional groups at each carbon position 2 3 4 5 6 7 8 2’ 3’ 4’ 5’ 6’ - - - OH - OH - - - OCH’ - - - - - OH R3 OH R3 - - OH - - - OH - OH - OH - - - OH - - - R11 OH - OCH3 - - - - OH - - - - - OH - OH - - - OH - - 7 2.3 Antimicrobial Plant Constituents Plants had developed biological system over the century to defend and survive from pathogens attack. This self-protective system in plant was partly depends on the plant secondary metabolites with antimicrobial properties. The most abundant secondary metabolites in plant are the polar phenolic compounds with great antioxidant properties (Dai & Mumper, 2010). 8 2.3.1 Chemical Properties of Leonurus sibiricus Leonurus sibiricus possess anticancer and antitumor properties (Satoh et al., 2003). Its aerial parts contain furanoditerpenelactones and four different diterpene-lactones namely, leonotinin, leonotin, dubiin and nepetaefuran. These compounds showed cytotoxic action on leukemia cells in tissue culture moderately (Satoh et al., 2003). L. sibiricus also exhibit allelopathic action against the surrounding plants (Mominul Islam & Kato-Noguchi, 2014). The allelopathic components in aqueous methanol extracts of L. sibiricus were able to interfere and even inhibit the germination and development of Italian ryegrass (Lolium multiflorum Lam.), garden cress (Lepidum sativum L.) and lettuce (Lactuca sativa L.) (Mominul Islam & Kato-Noguchi, 2014). The ascorbic acid and phenolic compounds in L. sibiricus give the antioxidants properties of this plant. In 100 g of the leaf powder, there are 8mg of ascorbic acids. The polyphenol contents of L. sibiricus ranges from 4-11mg for one gram of leaf sample depending on the drying temperature of the sample (Chua & Aminah, 2013). Antimicrobial properties are also found in L. sibiricus where 50 and 100 mg/mL of the ethanol extracts and 100 mg/mL of the water extracts of L. sibiricus were able to inhibit Staphylococcus aureus (Chua & Aminah, 2011). Effective inhibition of Aspergillus niger and Saccharomyces cerevisiae was observed when 25, 50 and 100mg/mL of the water extracts were applied to the media separately (Chua & Aminah, 2011). 9 2.3.2 Chemical Properties of Leonurus japonicus Antimicrobial properties of L. japonicus against bacteria and fungi had been discovered (Liang et al. 2013a; Yang et al., 2008). L. japonicus seeds contain two antimicrobial proteins namely, bornyl acetate and borneol that brought effective inhibition effects against bacteria (Alternaria alternata) and fungus (Ralstonia solanacearum) (Liang et al., 2013; Yang et al., 2008). The essential oils of L. japonicus also have antimicrobial activity (Liang et al, 2013a). L. japonicus mainly contain approximately 33% of diterpenes, 19% of phytone, 14% of phytol, 12% of caryophyllene oxide and 10% of β-caryophyleene and 13% of other less significant constituents (Liang et al, 2013a). The sesquiterpenes (phytol, phytone and caryophyllene) and diterpenes of L. japonicus had shown distinctive inhibition effects against Gram-positive bacteria. The sesquiterpene, β-caryophyleene affects the growth of Enterococcus faecium and E. faecalis (Liang et al, 2013a). Ethanol extracts of L. japonicus have the constituents that brought antibacterial activities against both Gram-positive bacteria and Gram-negative bacteria (Liang et al., 2013b). In the ethanol extract, the arteannuin B compounds able to inhibit Gram-negative bacteria (E. coli and Enterobacter aerogenes) with Minimal Inhibitory Concentration (MIC) values of 25µg/mL and 50µg/mL. Also in the ethanol extract, the chamigrenal compounds are able to inhibit Gram-positive bacteria (Macrococcus caseolyticus, Staphylococcus auricularis and Staphylococcus aureus with MIC values of 25µg/mL to 200µg/mL (Liang et al., 2013b) 10 2.4 Crop Pathogenic Fungi Crop pathogen is referred to the pathogenic microorganisms that influence and threaten the growth of vegetation. The resulted fungal diseases had brought numerous losses in crop production. In this study, six different crop pathogenic fungi were used in the inhibition test namely, Glomerella cingulata, Fusarium oxysporum, Fusarium solani, Pestalotiopsis sp., Colletotrichum sp. and Pyricularia oryzae. All of the stated fungi belong to phylum Ascomycota. 2.4.1 Glomerella cingulata Glomerella cingulata is a common crop pathogen in Malaysia that caused anthracnose disease on oil palms, rubbers and guavas (Snowdon, 2010). The infection symptoms always start with tiny dark lesion on the leaves and enlarge until the leaves turn black and shed (Wang et al., 2012). On the fruit, the symptoms are round, necrotic, sunken and red-bordered lesions on the fruit (Wang et al., 2012). 2.4.2 Fusarium oxysporum Fusarium oxysporum is a soil-borne pathogen that causes vascular wilt disease on a broad range of crops in Malaysia such as tomato, guava, banana, melon and whatnot (Dean et al, 2012). The infection interfere the vascular system of plants, so the plant will suffer from insufficient water supply. Therefore, the symptoms of vascular wilt disease on the infected 11 plants are stunted growth, wilting and yellowing leaves and brown ring is formed in the vascular bundle areas (Agrios, 2005). 2.4.3 Fusarium solani Fusarium solani is hardly differentiated from F. oxysporum by unprofessional mycologist but a trained mycologist can be identified F. solani by the presence of orange perithecia in pure culture (Burgess, Knight, Tesoriero & Hien, 2008). It is a soil borne fungus that cause serious crown, collar and root rots (Burgess, Knight, Tesoriero & Hien, 2008; Summerell et al., 2011). In Malaysia, this fungus usually infects the tropical fruits like papaya and banana (Zakariah, Chik, Kong & Salleh, 2012). F. solani is resistant towards most of the available fungicides (Summerell et al., 2011). 2.4.4 Pyricularia oryzae Pyricularia oryzae is a fungus that named differently at its different reproductive phase. At asexual reproduction stage, the fungus is known as Pyricularia oryzae (Agrios, 2005). The scientific name Magnaporthe oryzae is used at its sexual reproduction stage (Agrios, 2005). The species name “oryzae” is given based on its major infection on paddy that belongs to the genus Oryza. The infection will lead to the most destructive rice disease that known as rice blast which affects the paddy production of Malaysia (Dean et al, 2012). The infection form 12 lesions on the leaves with distinct diamond shaped lesions of white to grey colour and may enlarge which eventually destroy the entire leaves (Agrios, 2005). 2.4.5 Pestalotiopsis sp. Pestalotiopsis sp. affects only wounded or stressed plant especially on woody plant and conifer (Royal Horticulture Society, n.d.). This fungus causes leaf spots, petiole blights and bud rot of palm in certain cases (Elliott, 2005). In Malaysia, the common infected plants are rubbers, mangosteens, guavas and lychees (Snowdon, 2010). The infection symptoms are the steady enlarging tiny brown spots on leaves and the infected fruits turned harder with light pink colouration (Lim & Sangchote, 2003). 2.4.6 Colletotrichum sp. Colletotrichum sp. is known as Colletotrichum during anamorph and it is named differently during teleomorph which is Glomerella (Abd-Elsalam et al., 2010). It is the common fungi that cause anthracnose disease and rotting of many annual and perennial plants (Agrios, 2005). Malaysian commercial crops such as cereals, maize, corns, coffee, radish, papaya and rubber were usually infected by this fungus which brought significant losses in production (Dean et al., n.d.). 13 2.5 Diseases of Kacangma Plant diseases are normally caused by infection of pathogenic fungi, virus, bacterial, nematodes and infestation of insects. For pathogenic fungi, the spores spread and adhere on the plant parts. The spores are then germinate and develop by utilizing the plant nutrients. These will threaten the plant health and may cause dieback if infection was severe. The affected plant parts will show specific symptoms that can be an indication on the causal agent (Table 2.2). Fungi isolation from the affected plant parts and followed by morphological identification will allow the identification of the fungi up to the genus level based on their specific fruiting bodies structures. Then, molecular identification allows the identification up to the species level. Moreover, pathogenicity tests can be conducted to confirm the isolated fungus is the disease causal agent of the plant. Physiological effects on pathogenic fungi growth can be used to classify them into different groups. For example, every fungus has its own ideal temperature for growth and sporulation. Based on temperature factor, fungi are divided into three groups namely psychrophiles fungi that can grow optimally around 10oC, thermophiles fungi that can grow optimally around 40oC and mesophiles fungi that can grow optimally in between both psychrophiles and mesophiles preferred temperatures (Madan & Thind, 1998). With the knowledge on the minimum and maximum temperature limit that pathogenic fungi cannot thrive, post-harvest management on the kacangma products can be strategized. 14