Ganoderma Association with the Mortality of Acacia auriculiformis, Susceptibility to Different Hosts and Its Controls

The genus Ganoderma includes several wood decaying fungi on living trees as well as dead trunks and stumps, and has been recorded mostly in tropical and temperate countries. Generally, Ganoderma spp. cause extensive heart rots of standing trees by growing in the central, non-living woody tissues. Several studies have been carried out on Ganoderma diseases focusing on economic damage, severity of the disease and host range in many regions such as America, Asia, the Middle East and Europe [1]. Ganoderma lucidum has been reported as the causal organism of the heart rot disease of 91 hosts species Quercus spp. [2], Cocos nucifera [3], Camellia sinensis [4], Prunus persica [5], Vitis vinifera [6], Delonix regia and Cassia fistula. According to previous studies, several G. lucidum strains have been identified in the G. lucidum complex [7] having different host specificity. Control of root rot diseases is difficult as the pathogens survive on woody material in the soil. Green mould disease caused by Trichoderma spp. one of the serious problem of oyster mushroom and white button mushroom. It causes large economic losses to the mushroom growers. This was agreed by [8-11]. But this Trichoderma spp. has ability to control various plant diseases. This study was undertaken to examine the spread of root and stem rot disease in a particular study area, identify the causal agent of the disease and control the disease by means of biocontrol agent, Trichoderma. So, we can use this spent mushroom compost as a biofertilizer.


Introduction
The genus Ganoderma includes several wood decaying fungi on living trees as well as dead trunks and stumps, and has been recorded mostly in tropical and temperate countries. Generally, Ganoderma spp. cause extensive heart rots of standing trees by growing in the central, non-living woody tissues. Several studies have been carried out on Ganoderma diseases focusing on economic damage, severity of the disease and host range in many regions such as America, Asia, the Middle East and Europe [1]. Ganoderma lucidum has been reported as the causal organism of the heart rot disease of 91 hosts species Quercus spp. [2], Cocos nucifera [3], Camellia sinensis [4], Prunus persica [5], Vitis vinifera [6], Delonix regia and Cassia fistula. According to previous studies, several G. lucidum strains have been identified in the G. lucidum complex [7] having different host specificity. Control of root rot diseases is difficult as the pathogens survive on woody material in the soil. Green mould disease caused by Trichoderma spp. one of the serious problem of oyster mushroom and white button mushroom. It causes large economic losses to the mushroom growers. This was agreed by [8][9][10][11]. But this Trichoderma spp. has ability to control various plant diseases. This study was undertaken to examine the spread of root and stem rot disease in a particular study area, identify the causal agent of the disease and control the disease by means of biocontrol agent, Trichoderma. So, we can use this spent mushroom compost as a biofertilizer.

Materials and Methods
A study was carried out to calculate disease prevalence of Acacia auriculiformis at four selected different sites of Jahangirnagar University Campus, Bangladesh. Acacia auriculiformis are the dominant trees in every site of the campus. A total of fifty trees were randomly selected in each study area. Ganoderma spp. viz G. lucidum-1, G. lucidum-2, and G. lucidum-3 were collected from fully dead plants where as G. applanatum from partially dead trees. Symptomatology of infected trees due to Ganoderma was studied carefully.

Identification of Ganoderma spp. and Trichoderma spp.
In the present study, Ganoderma spp. was classified according to Corner [12] and Steyaert [13]. All of mycelium of four Ganoderma was hyaline, hyphae aseptate, basidiospores were thick walled, bitunicate, golden brown in color and ovate in shape. Colors of the colony of all wild Ganoderma were white ( Figure 1) The morphological, microscopic and cultural characteristic features of Ganoderma lucidum found more or less similar with previous researchers [1,14]. There was no chlamydospore found during present study as described by previous workers.

Disease severity index
The trees were scored for disease classes on a scale of 0 to 4 ( Table  1). The Disease Severity Index (DSI) was calculated using a modified method of Abdullah et al. [15] and Ilias [16].

Isolation of wild Ganoderma spp.
Both the pathogens and infested wood chips were cultured on PDA medium. Pieces (1 cm × 1 cm) of pileus and wood chips (1 cm × 1 cm) were placed at the center of the plate separately. Three replications for each isolate were maintained and incubated at 32 ± 2ºC. All of the isolates were pure cultured on PDA plates and stored at 4ºC until further use.

Morphological and cultural characteristics
Morphological Characterization of Ganoderma such as shape, size, thickness, margin, color, texture of pileus was examined. Microscopic studies were done by stereoscopic binocular microscope (OLYMPUS SZ 61, magnification 40X with Camera DP20, Japan). Hyphal features, colony characteristics were recorded.
T. koningii, T. viride (green strain), and T. viride (yellow strain) were collected from infected spent mushroom spawn packets of Pleurotus ostreatus, during December'2010 to February'2011. Antagonists Trichoderma harzianum was characterized according to Barnett [17] and Choi et al. [18] and. Others strain of Trichoderma were characterized as described by Barnett [17]. Four treatments combinations were used to assess susceptibility of Ganoderma. After cooling of saw dusts, fungal block each of 8 mm in size was inoculated into the test tubes containing sawdust under aseptic condition and cotton plugged. Test tubes were incubated at 32 ± 2ºC temperature. Radial growth of Ganoderma spp. on the test tube was measured at 10 days intervals and was analyzed statistically by MSTAT-C program.

Field trial
A field experiments were also conducted at Jahangirnagar University Campus during April to August 2011. A total of eight treatment combinations were used in field experiments ( Table 2). A total of fifty non infected plants were randomly selected in these purpose to inoculate the isolated Ganoderma to examine the capacity to infest on living host and the control of diseases prevalence using antagonists.

Mass culture of four Ganoderma isolates
The bamboo chips (3 cm) were sun dried for 10 days after cutting. Then these chips were mixed with wheat bran (30%), rice bean (30%) and 2% sugar solution with maintained 65% moisture. Then, these substrates were put into broad mouth test tubes (20 cm) and plugged with cotton. These test tubes were autoclaved for two times and waited until cooled. Test tubes were inoculated with each of four Ganoderma species. Data was collected until test tubes were filled with Ganoderma mycelium.

Preparation of Trichoderma spore suspension
At first, 20 ml of double distilled water was poured in each Petri dish having Trichoderma isolates. Then, each plate was scrapped to separate out mycelium of Trichoderma by using inoculating needle as mycelium of antagonist separated out. Then this solution was taken in plastic pot and covered with sterilized aluminum foil.
The stump or trunk region of Acacia sp. plants were holed by hammer and auger. Bamboo chips were inoculated into the plant by

Symptomatological study
Ganoderma infected plants showed symptoms with initially a bleached zone appeared in the wood which results delignification, drying of apical meristem or top dying of plants was the common symptoms on plants, wilting of plants, stem blackening, defoliation, white rot and root rot, loss of stiffness and finally, death of tree plants. All of the Ganoderma infected plants consisted at least 5 to 7 fruit bodies except Ganoderma applanatum where infected plants contained one fruit body and plant was defoliated. The symptoms recorded during study are in conformity with some researchers [14,19,20]. Dysfunctional xylem associated with large wounds on the roots which were thought to be due to delignification; caused by Ganoderma applanatum [21].

Calculation of Disease Severity Index (DSI)
The DSI value of Ganoderma at four selected sites of Jahangirnagar University Campus revealed that the highest disease incidence was found in site-1 (55.2%) in Acacia auriculiformis followed by site-2 (47.5%), site-4 (45%) and site-3 (36%) ( Table 3). Such findings partially supported by Nur and Abdullah [22] who cited the highest DSI (70.0%) due to Ganoderma infected oil palm seedlings. Disease progression of basal stem rot symptoms caused by Ganoderma boninense was recorded 100% in oil palm seedlings [23].

Assessment of wild Ganoderma (four) susceptibility to different host range
Ganoderma lucidum-1 was susceptible Acacia auriculiformis due to treatment T 2 which is statistically identical to T 4 followed by T 3 at 10 days (Table 4) but treatment T 4 exhibited more susceptible at 30 days. More or less similar results found in case of Ganoderma lucidum-2 and Ganoderma lucidum-3. Ganoderma applanatum showed no significant differences at 10 days but at 20 and 30 days only saw dusts showed more susceptible. In case of Artocarpus chaplasha, Ganoderma lucidum-1 was susceptible to treatment T 4 at 10, 20, 30 days respectively (Table  4). More or less similar patern recorded in case of Delonix regia (Table  5). Ganoderma applanatum showed better growth in sole saw dust of Delonix regia.
There was no clear pattern found in case of Albizia lebbeck for its growth (Table 4).
All of the Ganoderma spp. i.e., G. lucidum-1, G. lucidum-2, G. lucidum-3 and G. applanatum preferred to use saw dust of Mangifera indica followed by Ceriops decandra. This might be due to the presence of readily usable materials for its growth. In most of the cases, the least performance showed in Albizia procera and Dipterocarpus turbinatus might be the presence of secondary metabolites such as tannin, resins, and gums in wood. Root diseases caused by Ganoderma spp. seriously affected growth of Acacia spp. [24], Acacia auriculiformis and Acacia nilotica in India and Pakistan [25], Red rot disease of Acacia mangium caused by Ganoderma sp. reported in Malaysia [26]. Acacia auriculiformis affected by root rot disease caused by Ganoderma and Phellinus spp. in Papua New Guinea [27], heart rot disease caused by Ganoderma lucidum recorded in Quercus spp., Cocos nucifera, Camellia sinensis, Prunus persica, Vitis vinifera, Cassia nodosa, Casia fistula, Delonix regia, others 144 hosts in India [1].
Pathogenecity of Ganoderma spp. and antagonistic potentiality of Trichoderma spp.
In vivo pathogenecity of four Ganoderma spp. showed colonization of mycelium within 5 months, that means trees inoculated with bamboo chips of Ganoderma lucidum-1, G. lucidum-2, G. lucidum-3, G. applanatum showed mycelial run rate in the surroundings of inoculated area. In case of Ganoderma applanatum, mycelial run rate was just initiated (Figure 2). In vivo evaluation of potentiality of Trichoderma against Ganoderma spp. also studied. Bamboo chips of four Ganoderma spp. were inoculated with four selected Trichoderma spp. showed no mycelial run rate of Ganoderma spp., even Trichoderma spp. was sporulated over the inoculated area. Ganoderma applanatum as the most degradative wood colonizer [14]. The pathogenicity of Ganoderma spp. and the inhibitory effect of Trichoderma on Ganoderma spp. was followed earlier research [28].