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Nursery Screening for Ganoderma Response in Oil Palm Seedlings: A Manual. Techniques in Plantation Science

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This is a hands-on, practical guide covering seedling screening for disease response in oil palm for pathology, breeding and genetics. Oil palm is the top oil crop in the world and Ganoderma is the most devastating disease of oil palm. The authors are all actively engaged in oil palm seed production and breeding and bring together the many aspects of seedling disease testing in to one integrated manual. Presenting sound practices based on scientific innovation and knowledge, this guide provides techniques integrated with expertise and also looks towards future possibilities. Promoting green, eco-friendly agriculture, this book covers: Health and safety considerations; Media preparation for in vitro culture; Collecting isolates and culture preparation; Preparation of Ganoderma inoculum; Nursery inoculation; Scoring response. Based on experience and protocols, this is an invaluable manual for students and researchers in agriculture, plant breeders, growers, traders and production companies interested in the practicalities of oil palm pathology. It provides a resource for training, a knowledge base for people new to oil palm and a reference guide for managers, to ensure best practices in maximising sustainability and production of this important crop.

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2 Health and Safety Considerations

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Health and Safety Considerations

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Abstract

Standard and safety protocols are needed in all screening activities, both in the laboratory and in the nursery. The official standards can vary depending on country and local regulations, but good standards should be maintained whatever requirements are needed officially. The identification and elimination of hazards and risks, followed by developing specific safety procedures and procedures for preventing and responding to workplace accidents and injuries, are important features in establishing an effective occupational health and safety programme. Guidelines in health and safety issues relating to nursery screening of Ganoderma disease in oil palm are given below.

2.1  Health and Safety in the Laboratory

Various laboratory procedures are conducted in producing Ganoderma inoculum. These consist of isolating and culturing fungal isolates and preparing the inoculum. Therefore, good laboratory practices are required.

 

3 Media Preparation for In Vitro Culture of Ganoderma

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Media Preparation for In Vitro

Culture of Ganoderma

3

Abstract

The preparation of growth media is the first step in isolating any microorganism (bacteria and fungi) from the environment. Media may be solid or liquid. The culture medium formulation will affect the success of mycelium growth in culture. Although there are many culture media described for fungi, only a few of them have proven to be excellent in culturing

Ganoderma. This chapter explains how to prepare the different media used in culturing Ganoderma isolates that cause BSR disease. The methods provided here are for 1 l of medium (Tables 3.1 and 3.2), which may be scaled up or down depending on capacity and the volumes required; a table is provided giving the volumes of media needed with respect to the required numbers of cultures (see Chapter 5 of this manual, Table 5.5).

3.1  Water Agar Medium

Water agar (WA) is the simplest agar medium (Himedia, 2015). WA is often recommended as it is cheap and supports spore germination collected from

 

4 Collecting Ganoderma Isolates and Culture Preparation

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Collecting Ganoderma Isolates and Culture Preparation

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Abstract

Ganoderma may be isolated from the basidiocarp (the sporophore of

­basidiomycete), infected palm tissues (trunk or roots) or from single basidio­ spores; these sources produce abundant Ganoderma mycelia in culture

(Ho and Nawawi, 1986). Water agar (WA) or Ganoderma selective me­ dium (GSM) is used as the first growth medium; later, potato dextrose agar

(PDA) is used for rapid multiplication of Ganoderma cultures. The selec­ tion of samples especially from the basidiocarp can affect the success of

Ganoderma isolation; the use of younger basidiocarps is more favourable than older ones. An aseptic environment is needed for the successful isola­ tion and culturing of Ganoderma. Checklists of materials and equipment needed are provided in Tables 4.1 and 4.2.

4.1  Dikaryotic Mycelium Isolation from Basidiocarps

The use of young basidiocarps is preferred as they have more active mycelium than older basidiocarps. Also, the old basidiocarps have very thin dikary­ otic mycelium, which is harder to isolate (Fig. 4.1). The difference between young and old basidiocarps can be seen by their colour; old basidiocarps usually have a darker colour than young basidiocarps. Moreover, the ‘hat’ of old basidiocarps is harder and can show insects within, leading to more difficulty in isolation work.

 

5 Preparation of Ganoderma Inoculum

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5

Preparation of Ganoderma

Inoculum

Abstract

To conduct a nursery screen, a massive amount of Ganoderma inoculum is needed. This is used to inoculate/infect oil palm seedlings artificially. The success of nursery screening relies on a set of standardized parameters; one of them is the effectiveness of the inoculum source. Various parameters, such as the selection of aggressive isolates, material for the substrates, the volume of substrates and the length of fungus incubation, need to be standardized in order to provide robust and reproducible results. Currently, the use of rubber wood blocks as a substrate for Ganoderma is the most effective method of exposing and infecting seedlings with Ganoderma.

5.1  Preparation of Potato Agar Medium

In addition to the use of rubber wood blocks (RWBs) as a substrate, agar medium is also used as a starter food for Ganoderma to grow. This medium is added to the RWBs. It is different from potato dextrose agar (PDA), as potato is the only source of carbohydrate in potato agar (PA) and there are no antibiotics in this medium. The materials, equipment and tools required in preparing potato agar medium are given in Table 5.1.

 

6 Nursery Inoculation

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Nursery Inoculation

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Abstract

Parameters critical in the nursery for successful screening include: the level of shade; the inoculation stage; and the inoculation process as well as the trial conditions. Similar to the process in the laboratory, each parameter in the nursery should be as standardized as possible. However, variation in parameters will always occur. Therefore, standardization is aimed to minimize the variation between and within trials.

6.1  Nursery Preparation

Shade level is the most important parameter during the preparation of a nursery for Ganoderma screening (Fig. 6.1). Previous experiments have demonstrated that 90% shade gives the highest infection of the inoculated oil palm seedlings, probably because Ganoderma grows rapidly under dark conditions. The ambient temperature affects the success of seedling inoculation. Although Ganoderma depends on soil temperature, ambient air temperature has a significant impact on the temperature inside the nursery polybag. The materials, equipment and tools needed for nursery preparation are given in Table 6.1.

 

7 Scoring Response to Ganoderma

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Scoring Response to Ganoderma

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Abstract

To assess the variation in Ganoderma resistance, two different observations are conducted in the nursery. The scoring system of Ganoderma nursery screening consists of external and internal observations. External observations are conducted every 2 weeks (or possibly monthly), while the internal symptoms are observed at the end of each experiment. The appearance of

Ganoderma mycelium, sporophore and foliar symptoms in seedlings both alive and dead is counted as being infected by external observation. The seedlings are considered infected if, by internal observation, there are damaged tissues inside the bole and/or trunk.

7.1  External Observations

External observations follow the method described by Breton et al. (2006) and Idris et al. (2006). Foliar discoloration, indicating the appearance of fungus symptoms, is counted as an infection. Fungus symptoms can also appear as mycelium and/or basidiocarp (fruiting body). Standardization of symptom scoring is important in an effort to develop high-volume screening.

 

8 Future Possibilities

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Future Possibilities

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Abstract

It has been claimed that Ganoderma-resistant oil palm material holds the greatest hope for the future control of basal stem rot (BSR) in South-east

Asia. Conventional breeding currently requires germplasm with resistance and a phenotypic screening in both the nursery and the field. A better method might be provided by genotypic screening, which is independent of the environment and which can be exploited by breeders using markerassisted selection. The identification of genes and the development of DNA diagnostics for Ganoderma resistance would be a major step forward in developing disease-resistant oil palm. However, to develop this, it is first necessary to identify resistant genotypes.

8.1  Gene or Marker Detection for Ganoderma Partial

Resistance

The use of marker-assisted selection (MAS) using DNA markers is a useful tool in accelerating plant breeding (Forster et al., 2015). Both quantitative and qualitative traits can be analysed and selected using these techniques.

 

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