Fungi Associated with Root Rot of Pinus wallichiana Seedlings in Kashmir
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.
September 23, 2010; Accepted: February 22, 2011;
Published: May 06, 2011
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 Kochs postulate (Brock,
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.
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).
|| Root rot incidence of Blue pine seedlings in various districts
of Kashmir valley
|Values in parenthesis are arc sine transformed values
|| Isolation frequency (%) of root rot fungi from roots of blue
|*Percentage of colonies observed from 30 sampled root bits.
Values in parenthesis are arc sine transformed values
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 Kochs 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.
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