**2. Materials and methods**

#### **2.1 Forest health surveillance (FSH)**

Forest health surveillance (FHS), a formal and regular inspection of the planted forest, was performed in this study through the establishment of permanent sampling *New and Emerging Disease Threats to Forest Plantations in Sarawak Borneo, Malaysia DOI: http://dx.doi.org/10.5772/intechopen.107027*

plot (PSPs) surveillance and monitoring plots to assess the disease incidences and occurrences. FSH was undertaken between the period years 20152020, in various plantations selected randomly from the four forestry administration regions of Sarawak namely Miri, Bintulu, Sibu and Kuching (**Figure 1**). Field observations were carried out on forests planted with three major exotic tree species (**Table 1**).

We relied on information gathered by local foresters as well as our own observations to select the surveyed plantations in each studied forest region. Basically, the largest operational forest plantations were selected to better represent each forest region.

The plots were established by 30 30 m plot (the size of a plot varies in order to obtain a minimum of 100 living trees per plot) \* or alternatively by line transect with 10 planting rows and 10 trees per rows approach, which subsequently 100 trees will be assessed, assuming every tree were still alive (full stocking). All the trees assessed were labelled accordingly. GPS coordinates were recorded at every corner of the plot

**Figure 1.** *Map of Sarawak showing the distribution of forest tree plantation which are highlighted areas with red border.*


**Table 1.**

*Details of forest plantation species that were surveyed for disease pathogens.*

established and marked clearly with a peg. The plots were established at least 20 m from the boundary of plantation, roads or forest gaps. Details of plots are shown in **Table 2**.

#### **2.2 Disease assessment, sampling and disease incidence (DI)**

Trees with signs and symptoms such as the presence of any fungal fruiting bodies, swelling and cracking of the stems, branches and canopy appearances (i.e. wilting, yellowing, or defoliations) were observed and thoroughly recorded. Various observations were also taken, including the presence of animal damage, and appearances of insect/pests (i.e. termites' mounds, defoliators, borers or boreholes).

Samples of diseased leaves, branches, stems and roots were taken from diseased trees and collected for laboratory examination. The surface of cankers on stems was examined on the field using hand lenses for dark pycnidia [1] or any signs of fruiting bodies. Trees showing symptoms of branch death, wilting foliage, bark discolouration or sap exudation typical of Ceratocystis as well as showing signed of wood boring insect activities and sweet gaseous smell of pineapple were examined by chopping into the woody xylem at the base of the tree (most infections occur through the root system) or further up the stems for the characteristic xylem discolouration. Longitudinal strips of wood xylem (approximately 0.5 cm thick and around 10 to 15 cm long) were then cut from the discoloured xylem. In some cases, diseased trees were also felled and were cut into several sections measuring between 10 and 20 cm in length. The samples were then placed into paper bags before being transported to the Forest Pathology Laboratory, Industrial Forest Research Centre (IFRC), Kuching. The samples were stored at 4°C prior to fungal isolation.

Below ground variables were recorded for each tree on roots exposed to 20�50 cm from the base of the tree that exhibits symptoms of root rots. Variables recorded included are the presence or absence of any kind of root rot (red, brown, white) as well as the presence or absence of insect pests such as termites' tunnelling activities. Samples of insect pests were also collected for further identification.

The surveys aim to estimate the number of trees with any signs and symptoms of disease infection and thus determine the disease incidences (DI).

The DI were calculated by the following formula:

$$\text{DI} = \left(\frac{\mathfrak{n}}{\mathbf{N}}\right) \mathfrak{x} \mathbf{100\%.} \tag{1}$$

Where:

n = number of infected standing tree

N = total number of standing trees in one plot.

The disease incidences were then evaluated based on the indicator and rating as guided in **Table 3**. Values DI were reported in the results as ranges and are based on the plot data over the years of plot assessment.

#### **2.3 Fungal isolation and identification**

#### *2.3.1 Isolation of Ceratocystis wilt disease*

The carrot baiting method with minor modification was carried out to isolate Ceratocystis [3]. Pieces of the diseased stem were placed between two slices of fresh


*New and Emerging Disease Threats to Forest Plantations in Sarawak Borneo, Malaysia DOI: http://dx.doi.org/10.5772/intechopen.107027*


*<sup>\*</sup>Forestry Region (Figure 1): BTU, Bintulu; SBW, Sibu; KCH, Kuching; MY, Miri; R&D. \*\*Types of stand: R&D, Research and Development; CS, Commercial Stands.*

#### **Table 2.**

*Details of permanent sampling plots established and evaluated.*


#### **Table 3.**

*Indicator and ratings for evaluating the impact of forest health status in Sarawak [2].*

carrot and incubated in plastic containers that served as moisture chambers for 4–6 days. After 5–8 days, a pale orange ascospore mass appeared on the tip of the perithecia was then picked with a sterile syringe needle and transferred into a Petri dish plated with Potato Dextrose Agar medium and incubated at 26°C. All the isolates were identified based on the analyses of morphological characteristics [3–5]. The characteristics and structures of the culture, such as perithecium, ostiolar hyphae, ascospore and conidia (barrel-shaped and cylindrical-shaped) (μm) of all the fungal isolates, were measured and described. Morphological identification for this study was conducted by using a Leica compound microscope ICC50HD with a laptop workstation for data & image acquisition and a stereo microscope of Olympus SZ61 (SZ61TR-2X-DI-TP-118000A-SET) at Forest Pathology Laboratory, IFRC, Kuching.

*New and Emerging Disease Threats to Forest Plantations in Sarawak Borneo, Malaysia DOI: http://dx.doi.org/10.5772/intechopen.107027*

#### *2.3.2 Isolation of root rot disease*

Direct isolation was carried out to isolate the pathogen of root rot disease. Root tissues bearing disease symptoms were cut into several pieces approximately 3 mm 3 mm in size and were placed on malt extract agar (MEA) and incubated at room temperature for the fungus to grow. The cultures were then transferred to MEA for morphological studies.

#### *2.3.3 Isolation canker disease*

Segments of symptomatic plant parts were incubated in moist chambers for 2–3 days to induce the development of fruiting structures. These were then transferred to MEA and incubated at 25°C. Isolation from symptomatic tissue was also made directly onto MEA. Isolations were made onto MEA from fruiting structures occurring on cankered stems [6, 7].
