Abstract: This study was conducted to find out the cause of root rot disease of blue pine (Pinus wallichiana) seedlings which is an important limiting factor in its successful production right from the nursery raising. The pathogens associated with the disease were isolated, morphologically characterised and identified as Fusarium oxysporum f.sp. pini (Schlecht.) Synd. and Hans., Rhizoctonia solani Kuhn and Macrophomina phaseolina (Tassi) Goid. F. oxysporum was the most abundant pathogenic fungus in diseased roots of blue pine seedlings with isolation frequency of 38.6%, whereas R. solani and M. phaseolina showed isolation frequencies of 11.0 and 3.3%, respectively. All the three fungi proved pathogenic and caused characteristic root rot symptoms when inoculated separately. Root rot disease of Pinus wallichiana seedlings was prevalent in all the three districts of Kashmir valley surveyed, during 2008 and 2009.
INTRODUCTION
Pinus wallichiana (A.B. Jackson), the blue pine or Bhutan pine is an important conifer plant belongs to the family Pinaceae of order Coniferales. The blue pine is found in Himalayas at an elevation of 1500 to 3500 m a.m.s.l. In India the area under forests is estimated to be 639942 km2, with 45234 km2 under conifers alone. Of the total 20230 km2 area under forests in Jammu and Kashmir, conifers cover an area of 40.87% out of which 9.73% is occupied by blue pine (Anonymous, 2008). Blue pine is propagated mainly through nursery-raised seedlings. Pines, like most of the forest trees, face severe problems in successful regeneration under existing natural conditions. Forest trees are often exposed to persistent pathogenic attacks, particularly those causing root rots. Among the conifers, root rot fungal pathogens have mostly been encountered on pines followed by deodar (Bhardwaj et al., 1988). The important root rot fungi posing serious threat to forest nurseries include the species of Fusarium, Phytophthora, Pythium, Rhizoctonia, Macrophomina and Cylindrocladium (Gupta et al., 1988; Huang and Kuhlman, 1990; Asiegbu et al., 1999). These pathogens often invade terminal unsuberized roots of young seedlings and cause late damping off or root rot thereby kill the host. The fungi penetrate into root epidermal cell wall, grow inter-cellularly, decompose cell wall materials and persist by metabolising cell contents (Garret, 1970). Root rot incidence/severity reduces the dry matter production and partitioning in affected plants (Aigbe and Remison, 2010). In present study Fusarium oxysporum, Rhizoctonia solani and Macrophomina phaseolina were found responsible for the root rot of Pinus wallichiana seedlings under temperate agroclimatic conditions of Kashmir. Fusarium spp. are well known forest nursery pathogens causing root rot in older seedlings as well as pre and post emergence damping-off, thus reducing seed germination and seedling survival (Machon et al., 2009). F. Oxysporum is the most important potential root pathogen in forest nurseries and is commonly associated with diseased conifer seedlings (Allin et al., 2004; Enebak et al., 1990). Rhizoctonia spp. are highly virulent pathogens of forest nurseries that can cause damping-off and root rot in seedlings (Chakravarty and Mishra, 2007). Macrophomina phaseolina is one of the most destructive plant pathogens that is both root and soil inhabiting and has a wide host range affecting more than 500 plant species including pines (Kadliesko, 1994). To increase biodiversity and to reduce soil erosion risk, besides revitalizing the rural economy and provide employment, has resulted an increase in the production of forest nursery seedlings. The early development of out planted seedlings in the field depends to a large extent on their physiological condition in the nursery. As such production of healthy seedlings is one of the main prerequisites for successful reforestation.
MATERIALS AND METHODS
During April-May 2008 and 2009, nine pine growing nurseries were surveyed from the three districts viz., Anantnag, Srinagar and Baramulla of Kashmir valley to assess the status of the disease. Three major nurseries were selected in each district. In each nursery six plots of 1x2 m size each were randomly selected, to assess the root rot incidence in one year old blue pine seedlings. Total number of seedlings examined and the number of seedlings showing root rot symptoms were recorded from each plot and percent disease incidence calculated by using the formula:
Ten plants from each surveyed nursery showing typical root rot/wilt symptoms were taken along with soil, bagged separately in polythene envelopes and immediately brought to the laboratory. The seedlings were then carefully uprooted and observations on deviated morphology of plant parts recorded. The seedlings with needle discolouration and stunted growth, following excavation also showed symptoms of root rot. The infected root portions were washed in running tap water. From each root system three to five main lateral roots showing decay symptoms were selected randomly. From each selected root, single segment of 5 mm in length was cut at the zone of advancing decay. The bits were surface sterilized in 0.1% mercuric chloride for one minute followed by three consecutive rinses in sterilized deionised water. The bits were blotter dried and then aseptically transferred to Potato Dextrose Agar (PDA) medium in sterile Petri-plates and incubated at 25±2°C. The plates were observed regularly and outgrowing mycelia were sub-cultured immediately. The cultures were purified by hyphal tip method (Dasgupta, 1988). Various cultural and morphological characteristics of isolated fungi were recorded by making visual and microscopic observations and compared with standard descriptions given by Nelson et al. (1983) and Sneh et al. (1991). The pathogenicity tests of root rot fungi isolated from the roots of diseased blue pine seedlings were carried out separately by following the Koch’s postulate (Brock, 2000).
For the determination of isolation frequencies of root rot pathogens, three segments per root system were selected from ten randomly selected plants of each site. The number of isolates of fungal pathogens isolated from sampled root segments was determined and the mean isolation frequency rates were calculated as the number of isolates of selected fungus from 30 sampled root bits.
RESULTS
To find out the status of root rot disease in blue pine (Pinus wallichiana) seedlings in Kashmir valley, various forest nurseries were surveyed during two consecutive years (2008 and 2009) in the month of May. The data presented in Table 1 revealed that mean disease incidence at the locations surveyed varied from 18.8 to 44.7% with overall mean incidence of 30.8%. Comparison of year-wise data revealed more disease prevalence (32.3%) during 2009 as compared to 2008 (29.4%). The degree of disease incidence varied from 17.2 to 43.2% and 20.4 to 46.2% in the year 2008 and 2009, respectively.
Isolations made from diseased roots of blue pine seedlings, collected during survey yielded three root rot fungal pathogens including Fusarium oxysporum f.sp. pini Schlecht., Rhizoctonia solani Kuhn and Macrophomina phaseolina (Tassi) Goid. Besides these, some saprophytic fungi viz., Mucor, Rhizophus, Penicillium, Aspergillus and Trichoderma spp. were also observed on affected root portions. The data on the isolation frequencies of root rot fungi revealed that F. oxysporum was isolated from all the surveyed nurseries with highest isolation frequency (38.6%), followed by R. solani (11.0%). M. phaseolina was occasionally isolated from sampled root bits with isolation frequency of 3.3% (Table 2).
| Table 1: | Root rot incidence of Blue pine seedlings in various districts of Kashmir valley |
| Values in parenthesis are arc sine transformed values | |
| Table 2: | Isolation frequency (%) of root rot fungi from roots of blue pine seedlings |
| *Percentage of colonies observed from 30 sampled root bits. Values in parenthesis are arc sine transformed values | |
DISCUSSION
Root rot disease caused by soil borne fungi is a serious problem faced by nursery growers throughout the world. Assessment of the status of a disease is one of the pre-requisites for devising suitable disease management strategies. The survey of forest nurseries of Kashmir valley revealed that root rot in blue pine seedlings was more or less prevalent in all the locations surveyed. The disease incidence varied from location to location. The disease incidence was significantly higher in the year 2009 which may be attributed to more precipitation (84.61 mm) and high temperature (23.53°C) prevailing during 2009 in comparison to 2008 (65.00 mm and 15.74°C, respectively). High moisture content (40-60%) in growth substrate generally favours the growth and infection by root rot pathogens of conifer seedlings. Bloomberg (1981) also reported that warm weather (25-35°C) favours the pathogen to induce losses and predispose the host to infection by the pathogen.
On the basis of morphological characters, pathogenicity and comparison with the authentic descriptions, the pathogens isolated were identified as Fusarium oxysporum f.sp. pini Schlecht. Synd and Hans., Rhizoctonia solani Kuhn. and Macrophomina phaseolina (Tassi) Goid. These pathogens have been reported elsewhere as causal organisms of root rot of conifer seedlings other than blue pine from Wisconsin (USA), Uppsala Sweden and Ontario forest nurseries (Bloomberg, 1981; Ocamb et al., 2002; Landis, 1999; Stepniewska-Jarosz et al., 2006; Pinto et al., 2006; Zakeri et al., 2010). The pathogenicity of isolated root rot fungi was established by proving Koch’s postulates. The symptoms characterised to root rot were the needle discolouration, stunted growth and tip dieback. Root system showed lack of laterals and extensive cortical decay leading to the ultimate death of roots.
Observations recorded on isolation frequencies of root rot pathogens demonstrated that different fungi colonized the affected roots of blue pine seedlings in forest nurseries. The most frequently isolated fungal pathogens were F. oxysporum, R. solani and M. phaseolina. Some saprophytic fungi viz., Rhizopus, Mucor, Penicillium, Aspergillus and Trichoderma sp., were also isolated from diseased pine roots. F. oxysporum was the most frequently encountered pathogen in diseased roots of blue pine seedlings, with highest average isolation frequency of 38.6%. The isolation frequency of R. solani was 11.0% while as M. phaseolina was occasionally isolated with isolation frequency of 3.3%. These results corroborates the findings of Menkis et al. (2006), who recorded 44.6% isolation frequency of F. oxysporum from colonized roots of Pinus sylvestris seedlings and 0.3% isolation frequency of R. solani from the diseased roots of Picea abies seedlings from Uppsala Sweden. Fusarium root rot of forest nursery seedlings is most often attributed to the fungus Fusarium oxysporum, although some other Fusaria have sometimes been implicated in root rot of conifer nursery seedlings (Enebak et al., 1990; Juzwik and Rugg, 1996). Barbara and Leszek, (2004) observed that R. solani is responsible for damping-off and root rot in young pine seedlings. Barnard et al., (1994) also reported that M. phaseolina caused charcoal root rot and is a factor in the development of black root rot together with Fusarium spp. in some forest tree nurseries. In conclusion root rot of blue pine seedlings was observed at moderate to high levels in all the nurseries surveyed and three most potential pathogens were found responsible, there by poses a serious threat to the regeneration of pine plantation. As such control measures that are devised for root rot must be taken into account in order to meet the demand of conifer nursery seedlings by producing more healthy seedlings.