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Research Article
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Management of Root Rot Caused by Rhizoctonia solani and Fusarium oxysporum in Blue Pine (Pinus wallichiana) Through use of Fungal Antagonists |
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Gh. Hassan Dar,
M.A. Beig,
F.A. Ahanger,
Nadeem A. Ganai
and
M. Ashraf Ahangar
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ABSTRACT
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The present study was aimed to identify root rot pathogens of blue pine (Pinus wallichiana) in Kashmir and develop appropriate eco-friendly disease management strategy. During nursery surveys, Fusarium oxysporum, Rhizoctonia solani and Macrophomina phaseolina were found root rot incitants with isolation frequency of 47.3, 29.7 and 13.0%, respectively. Locally isolated antagonists inflicted 33.0-73.3 and 29.5-70.8% mycelial growth inhibition in F. oxysporum and R. solani, respectively, with Trichoderma harzianum and T. viride proving most effective. The mycorrhizal fungi, Pisolithus tinctorius and Laccaria laccata, significantly inhibited the growth of R. solani and F. oxysporum by 46.2 and 45.4 and 44.7 and 43.7%, respectively. Bioagents significantly improved seedling biomass and root/shoot length. Mycorrhizal plants showed 5-13 fold higher rhizosphere phosphatase activity than non-mycorrhizal ones. Four effective fungal bioagents, inoculated individually and in combination with pathogen under nursery conditions, significantly improved seedling biomass and height with maximum gain by P. tinctorius and L. laccata. Rhizoctonia infection decreased biomass and seedling height by 32.6 and 35.4%, whereas bioagents mitigated the pathogenic effect. The bioagents in R. solani/F. oxysporum-infected soil significantly improved seedling biomass and height over pathogen treatments alone. P. tinctorius and L. laccata exhibited 44.2 and 39.1% root colonization in comparison to 19.5-24.2% in presence of pathogens. The study revealed that bioagents, especially mycorrhizae, effectively mitigate root rot in blue pine and can be efficiently exploited in integrated disease management module.
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How
to cite this article:
Gh. Hassan Dar, M.A. Beig, F.A. Ahanger, Nadeem A. Ganai and M. Ashraf Ahangar, 2011. Management of Root Rot Caused by Rhizoctonia solani and Fusarium oxysporum in Blue Pine (Pinus wallichiana) Through use of Fungal Antagonists. Asian Journal of Plant Pathology, 5: 62-74.
URL: https://scialert.net/abstract/?doi=ajppaj.2011.62.74
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Received: March 24, 2011;
Accepted: May 18, 2011;
Published: July 12, 2011
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INTRODUCTION
The Himalayan mountainous ranges harbour four out of the six indigenous pine
species of Indian subcontinent, viz., Pinus wallichiana, P. roxburghii, P.
gerardiana and P. kesiya. Of these, blue pine (Pinus wallichiana
Jack.) has its native habitat spread over Eastern Afghanistan, South-eastern
Tibet and China to North Burma and is mostly found in Himalayas at an elevation
of 1500 to 3500 m m.a.s.l. The tree is widely distributed in Jammu and Kashmir,
Himachal Pradesh and Gharwal hills in the West, spreading towards Bhutan with
sporadic distribution in Arunachal Pradesh in the east. Of the total 20,230
km2 area under forests in Jammu and Kashmir State, conifers cover
40.87% area out of which blue pines occupy 9.73% area (Beig
et al., 2008a).
Blue pine, locally known as Kail pine, faces several constraints in its successful
regeneration in field. The plants are often exposed to persistent pathogenic
attacks, particularly those inciting root rot and wilt diseases, at primary
stages of plant establishment. Root rot, wilt and die-back on container-grown
conifers such as spruce and pine is major problem since 1990s in Europe
(Lilja et al., 2010). The root rot fungi which
pose serious threat to forest nurseries include the species of Fusarium,
Phytophthora, Pythium, Rhizoctonia, Macrophomina and Cylindrocladium
(Huang and Kuhlman, 1990; Asiegbu
et al., 1999; Wafaa and Haggag, 2002). These
pathogens often invade terminal unsuberized roots of young seedlings and cause
late damping off or root rot/wilt thereby kill the host. The fungi penetrate
into root epidermal cell wall, grow intercellularly, decompose cell wall constituents
and persist by metabolising cell contents. Root rot is world-wide a serious
problem in pine seedlings and serious losses due to this disease have been reported
from Ontario, Canada, USA and many European countries (Greifenghagen
et al., 1991; Lilja and Rikala, 2000). In
view of highly devastating nature of root rot pathogens, effective disease management
is essential to raise healthy pine seedlings for successful implementation of
reforestation and afforestation programmes.
Several approaches involving fungicide use and cultural measures have been
adopted by the nursery growers to reduce the root rot incidence; yet the disease
continues to assume serious threat to conifers (Shah et
al., 1999). The use of biocontrol agents is presently gaining momentum
as a supplement to chemical treatment in integrated disease management module.
The effective use of antagonistic bacteria, actinomycetes and fungi as biocontrol
agents against several soil-borne pathogens have been demonstrated in several
field and horticultural crops (Yobo et al., 2010).
The free-living rhizosphere micro-organisms such as Trichoderma, Gliocladium,
Penicillium, Pseudomonas, Bacillus, Azotobacter, Azospirillum, etc.,
favourably influence the plant growth directly or indirectly; whereas mycotrophy
benefits the host plant by enhancing the root capacity to absorb nutrients,
extracting the nutrients beyond depletion zones in soil and protecting the root
from pathogenic invasions (Eziashi et al., 2006;
Gacitua et al., 2009; Lu
and Huang, 2010; Heydari and Pessarakli, 2010).
The fungal antagonists may compete for an ecological niche by consuming available
nutrients and by secreting a spectrum of biochemicals effective against various
fungal pathogens. These biochemicals may include cell wall-degrading enzymes,
siderophores, chelating iron, a wide variety of volatile and non-volatile antibiotics,
etc. (Susi et al., 2011). Widyastuti
et al. (2003) and Benitez et al. (2004)
speculated that indirect and direct defensive mechanisms of Trichoderma
species act coordinately. Accordingly the success in biocontrol process depends
on the antagonisitic strain involved, the antagonized fungus, the crop plant
and the environmental conditions, including nutrient availability, pH, temperature
and iron concentration. Root drench of potted Pinus radiata with Trichoderma
atroviride isolate R32 not only enhanced seedling root biomass and stem
diameter but also induced systemic resistance to Diplodia pinea and reduced
dieback incidence by 20% compared to untreated controls (Reglinski
et al., 2011).
Symbionts, such as mycorrhizal fungi and free living organisms form integral
components of pine rhizosphere, an area exhibiting all kinds of antagonistic,
parasitic and growth promoting interactions (Taylor and
Alexander, 2005). Mycorrhiza reportedly aid young seedlings in their early
survival and establishment through intricate and complex system of hyphal networks
thereby not only ensures the sustained nutrient supply but also provides protection
against invading pathogens (Podila and Douds, 2001; Jha
et al., 2008). The culture filtrate of mycorrhiza, Suillus cdlinius
Hebeloma mesophaeum and Paxillus sp., reportedly exhibit antagonistic
effect on the mycelial growth and spore germination of Fusarium oxysporum
and Pythium vexans and the antifungal activity has been attributed
to oxalic acid production (Quarraqi et al., 2005;
Yamaji et al., 2005). Inoculation of Pisolithus
tinctorius in Pinus densiflora significantly increased dry matter
and stem diameter when compared to non-inoculated seedlings (Choi
et al., 2005). Paxillus involutus effectively controlled root
rot caused by Fusarium oxysporum and Fusarium moniliforme in red
pine (Pal and Gardener, 2006). Efforts are underway to
improve the quality of forest nursery seedlings through inoculation of suitable
mycorrhizal strain in association with other compatible bioagents.
The perusal of literature reveals that some work has been done on the management
of root rot diseases in conifers other than blue pine (Enebak
et al., 1990). From India, meagre information about the causal pathogens
responsible, symptomology and management of diseases is available for conifers
(Kaushik et al., 2002; Bisht
et al., 2003). In view of the importance of root rot disease in blue
pine and the damage inflicted by it, there is urgent need to develop a suitable
disease management strategy which may include biological control as an important
component. Therefore, the present investigation was undertaken to assess the
bio-efficacy of fungal antagonists in the management of root rot disease on
blue pine under temperate conditions of Kashmir.
MATERIALS AND METHODS
The root rot pathogens were isolated from diseased roots, collected during
the survey of blue pine (Pinus wallichiana) nurseries in years 2007 and
2008. Three to four surface-sterilized diseased root bits of 3-5 mm size were
aseptically transferred to Potato Dextrose Agar (PDA) medium and plates incubated
at 25±2oC for mycelial growth. The cultures were purified
by hyphal tip method (Dasgupta, 1988). Various cultural
and morphological characteristics of isolated fungi were recorded by visual
and microscopic examinations. Morphological characteristics of isolated fungi
were compared with standard descriptions given by Nelson et
al. (1983) and Sneh et al. (1998). The
fungal/bacterial antagonists and ectomycorrhiza were isolated from the rhizosphere
of blue pine trees by dilution plate method on PDA and Modified Melin Norkans
agar (MMN) media, respectively (Marx, 1969; Rangeshwaran
and Prasad, 2000). The cultures were purified by single spore/hyphal tip
method. The identification of isolated fungi was done on the basis of cultural
and morphological characteristics (Arx, 1981). The isolated
ectomycorrhiza were identified on the basis of culturo-morphological characteristics
and identified with the help of standard descriptions (Godbout
and Fortin, 1985; Lakhanpal, 1988).
In vitro assessment of biocontrol agents: The antagonistic activity
of isolated rhizosphere fungi/bacterium and mycorrhizae against root rot pathogens
was assessed by dual culture technique using PDA and MMN agar media, respectively
(Dhingra and Sinclair, 1985). Treatments were replicated
four times in a completely randomized design and incubated at 25±2°C
for 8 days. Radial growth was recorded on 8th day of incubation and mycelial
inhibition calculated as per Vincent (1947).
The morphology of hyphae in interaction zone was observed under light (10x)
microscope. Clear and characteristically parasitized hyphae were examined under
high magnification power (40x). Based on the growth and mycoparasitic nature,
biocontrol agents were grouped into various categories as per the scale given
byMunshi and Dar (2004). The antifungal activity of
bacterial antagonist was assessed by dual culture technique according to the
method given by Dennis and Webster (1971), using PDA
medium. Control plates inoculated with pathogen alone were also maintained.
The treatment plates, replicated four times, were incubated at 28±2°C
in completely randomized design. Mycelial growth of pathogen and inhibition
zone was measured after 72 h of incubation.
In vivo evaluation of antagonists: The efficacy of antagonist
under in vivo conditions was assessed as per the method described earlier
(Ahangar et al., 2011). The treatments were replicated
10 times and arranged in a completely randomized block design in a glasshouse
at 26±3°C. The seedlings were gently uprooted after 3 months to assess
fresh plant biomass and mycorrhizal development. The entire root systems were
examined under a stereomicroscope to count the number of mycorrhizal short roots
(Daughtridge et al., 1986). The mycorrhizal colonization
was assessed by counting the total number of mycorrhizal short roots formed
by inoculated fungi against the total number of short roots observed. Root and
shoot length of seedlings was measured and alkaline acid phosphatase activity
of rhizosphere soil was estimated as per the method of Tabatabai
and Bremner (1969). The amount of ρ-nitrophenol (PNP) released was
calculated in reference to the standard curve prepared by using different concentrations
of PNP. The enzyme activity was expressed in terms of μM PNP released g-1
soil h-1.
Nursery studies on pathogen-antagonist interaction: The most effective
five fungal antagonists, including two ectomycorrhiza, were evaluated individually
and in combination with major pathogens. The pathogenic inoculums of non-mycorrhizal
fungus were incorporated @ 10 g kg-1 mixture to ensure sick soil
formation. Biocontrol agents, multiplied on wheat bran, were added @ 10 g kg-1
potting mixture (with an inoculum load of 1x109 cfu g-1)
as per treatment. Ectomycorrhiza were multiplied in vermiculite-peat moss based
carrier added to sick soil @ 15 mL bag-1. The antagonist additions
to sick soil were made 15 days before seed sowing. Non-sick soil and sick soils
without antagonist inoculation served as controls. The surface sterilized and
stratified healthy seeds of blue pine were sown 3 per bag of sterilized potting
mixture of one kg in a 1.5 kg capacity plastics bag. After germination seedlings
were thinned out to one per bag. Each treatment replicated 10 times was arranged
in a CRD in a greenhouse at 25±3°C and irrigated with sterile water
as and when required. No fertilizers or protective chemicals were applied throughout
the study. Ectomycorrhizal infection in roots was estimated and roots examined
under a stereomicroscope to count the number of mycorrhizal short roots as suggested
by Beckjord et al. (1984).
The fresh plant biomass and seedling height were estimated 90 and 180 days
after seedling emergence. Root colour intensity and root rot index was measured
on the basis of root area affected according to the root rot index rating scale
described by Purkayastha et al. (1981). The data
were subject to analysis of variance and means compared using Duncans
new multiple range test (Gomez and Gomez, 1984). Arcsine
and square transformations were employed wherever applicable. The data analysis
was carried out using Statistical Packages for Social Sciences (SPSS ver. 11.5
Chicago USA for windows).
RESULTS AND DISCUSSION
Root rot pathogens of pines: During preliminary survey the roots of
blue pine seedling showing root rot and wilt symptoms, collected from forest
nurseries in Anantnag and Baramulla districts of Jammu and Kashmir, were found
infected by three root rot causing fungi viz., Fusarium oxysporum f.sp.
pini Schlecht. Synd. and Hans., Rhizoctonia solani Kuhn. and Macrophomina
phaseolina (Tassi.) Goid. Besides these, some saprophytic fungi were isolated
from the affected root portions. Fusarium oxysporum was isolated from
all the surveyed nurseries with an overall isolation frequency of 47.3% which
was followed by Rhizoctonia solani and Macrophomina phaseolina
with overall isolation frequencies of 29.7 and 13.0%, respectively and the rest
10% were the species of Mucor, Rhizophus, Penicillium, Aspergillus and
Trichoderma. These observations are in line with Pinto
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. Lilja et al. (1995)
and Stepniewska-Jarosz et al. (2006) frequently
isolated the above pathogens from diseased conifer roots, cone scales and seeds
of Scots pine from Florida and Poland forest nurseries, respectively. Fusarium
species, especially F. oxysporum, reportedly is highly potential
root rot pathogens in many forest nurseries (Enebak et
al., 1990).
Antagonistic isolates: Three non-mycorrhizal fungal antagonists Trichoderma
viride, T. harzianum and Gliocladium roseum and four mycorrhizal
fungi Pisolithus tinctorius, Laccaria laccata, Boletus spp. and
Suillus granulatus were isolated from blue pine rhizosphere. Their identification
was made on the basis of morpho-cultural characteristics. L. laccata
and S. granulatus have previously been isolated from the basidiomata
collected from Pinus patula plantations by Reddy
and Natarajan (1997) from Nilgiri hills, Tamil Nadu, India and Yamada
et al. (2001) from Ibaraki, Japan. Dar et
al. (2009) have reported the presence of Pisolithus tinctorius,
Laccaria laccata, Boletus spp. and Suillus granulatus from
Gulmarg, Bandipora and Baramulla conifer forests of Kashmir with high rhizosphere
phosphatase activity and root colonizing potential.
Pathogenic growth inhibition by antagonists: In vitro evaluation
of antagonists against F. oxysporum and R. solani in dual culture
revealed that all the tested biocontrol agents significantly inhibited the mycelial
growth of pathogens (Table 1). The mycelial inhibition on
8th day ranged from 33.0 to 73.3% and 29.5 to 70.8% for F. oxysporum
and R. solani, respectively, with T. harzianum proving more effective
against both the pathogens, followed by T. viride (70.5 and 64.8%) and
Gliocladium roseum (50.0 and 54.7%). Trichoderma species proved highly
antagonistic and exhibited strong mycoparasitic activity.
Table 1: |
In vitro effect of various antagonists on mycelial
growth of Fusarium oxysporum and Rhizoctonia solani in dual
culture |
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HA: Highly antagonistic, MA: Moderately antagonistic, A: Antagonistic, SA: Slow antagonistic, * +: Inhibition zone present, -: inhibition zone absent |
They completely overgrew the host mycelia once in contact with pathogens and
formed hyphal coils on pathogenic colonies. G. roseum and Pseudomonas
fluorescens grew slowly and developed zone of inhibition against the pathogen.
Amongst the mycorrhizal fungi Pisolithus tinctorius and Laccaria laccata
inflicted significantly higher mycelial growth inhibition of 45.4 and 46.2 and
43.7 and 44.7% in F. oxysporum and R. solani, respectively. Further,
L. laccata and P. tintorius were observed to be moderate antagonists
as they caused rupture and twisting of pathogenic hypha followed by their gradual
desiccation, protoplasm shrinkage and ultimately cell lysis. Boletus spp.
and Suillus granulatus inflicted only 33.0 and 29.5 and 33.7 and 36.4%
inhibition in mycelial growth of F. oxysporum and R. solani, respectively,
so were categorized as slow antagonists. The growth inhibitive effects of antagonists
are in agreement with Rudresh et al. (2005) who
observed 72.1 and 77.0% growth inhibition in R. solani and F. oxysporum,
respectively, by T. harzianum and T. viride which also exhibited
strong mycoparasitic activity and completely overgrew the host mycelia once
in contact with them. Dubey (1998) observed hyphal coil
or hook or appresoria formation by T. harzianum on fungal colony of Rhizoctonia
solani. Trichoderma species reportedly produce chitinase and β1-3,
glucanase enzymes which degrade cell wall and cause hyphal lysis of pathogens
(Wu et al., 1986).
The formation of inhibition zone by T. viride, G. roseum and P. fluorescens
suggests the involvement of strong antibiosis mechanism, possibly due to the
production of volatile metabolites and diffusible chemicals produced by antagonist.
Munshi and Dar (2004) noticed inhibition zone formation
by Gliocladium sp. against Fusarium pallidoroseum. The mycoparasitic
activity of Laccaria laccata against R. solani and Fusarium
sp. has earlier been suggested by Zhao and Kuo (1988).
Plant growth improvement by antagonists: Preliminary in vivo evaluation
of mycorrhizal and non-mycorrhizal fungal bioagents in improving pine seedling
growth was assessed in pot culture experiments. All the test biocontrol agents
significantly improved root and shoot length of pine seedlings and yielded higher
biomass than uninoculated control, as observed 90 days after seedling emergence
(Table 2). L. laccata and P. tinctorius depicted
significantly higher fresh plant biomass of 0.99 and 0.97 g plant-1,
respectively, followed by T. harzianum (0.85 g plant-1) and
T. viride (0.83 g plant-1) in comparison to un-inoculated
control (0.62 g plant-1).
Table 2: |
In vivo effect of fungal antagonists, including ectomycorrhiza,
on plant growth, root colonization and rhizosphere acid phosphatase activity
in blue pine seedlings |
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Observations taken 3 months after inoculation |
Similar trend was noticed in case of root and shoot length with significantly
higher length of 10.5 and 9.4 cm, respectively, in L. laccata in comparison
to the respective values of 5.9 and 6.3 cm in un-inoculated control. This
was followed P. tinctorius, T. harzianum and T. viride. The findings
are in agreement with Villeneuve et al. (1991)
who observed 40% growth increase in Douglas-fir seedlings due to the inoculation
of L. laccata and Reddy and Natarajan (1997)
who reported 38.0 and 186.5% increase in seedling height and shoot dry weight
of Pinus patula due to L. laccata inoculation after 8 months.
It appears that mycorrhizal fungi imparted protection, induced resistance or
released antimicrobial compounds to favour plant growth. Fungal sheath around
the roots seemed to have restricted the fungal advancement into mycorrhizal
cortex. Protection of conifer seedlings against Fusarium spp. due to
L. laccata has been attributed to the possible production of antifungal
phenol compounds by the host in presence of mycorrhizal species (Chakravarty
et al., 1991). Farquhar and Peterson (1990)
showed that Pinus resinosa seedlings inoculated with Paxillus involutus
had induced resistance to F. oxysporum.
Mycorrhizal colonization and rhizosphere phosphatase activity: The mycorrhizal
root colonization and rhizosphere phosphatase activity was higher in mycorrhiza
inoculated treatments. The tested ectomycorrhizal fungi colonized 33.9-42.0%
roots in 90 days, developed symbiotic association with pine seedling roots and
improved mycorrhizal short root formation (Table 2). Significantly
high mycorrhizal root colonization was observed in pine inoculated with L.
laccata (42.0%) with better rhizosphere phosphatase activity of 212.0 μM
PNP g-1 soil. However, P. tinctorius was at par with L.
laccata with root colonization of 40.5% and rhizosphere phosphatase activity
of 214.5 μM PNP g-1 soil. In case of soils inoculated with non-mycorrhizal
bioagents and untreated control the phosphatase activity was significantly very
low ranging from 16.0 to 42.0 μM PNP g-1 soil. Mycorrhizal plants
exhibited 5-13 fold higher rhizosphere phosphatase activity than non-mycorrhizal
ones. Acid phosphatase is solely of extracellular origin and is involved in
the mineralization of organic phosphates. The phosphatase enzymes solubilize
insoluble forms of phosphorus and other nutrients not readily available to plant
roots. Dunabeitia et al. (2004) and Beig
et al. (2008b) observed greater acid phosphatase activity in the
rhizosphere of mycorrhizal plants than non-mycorrhizal ones. T. harzianum
has also the ability to solubilize many plant nutrients including rock phosphate
from their solid phase compounds by their enzymatic activity (Altomare
et al, 1999). The synergistic effect of biocontrol agents in combined
inoculation resulted higher phosphatase activity in the rhizosphere of blue
pine seedlings.
Plant growth improvement in presence of pathogens: Under field nursery conditions four effective fungal biocontrol agents including 2 effective ectomycorrhiza were inoculated individually and in combination with root rot pathogen (F. oxysporum or R. solani). All the test bioagents significantly improved plant growth in terms of biomass and seedling height in blue pine whereas presence of root-rot pathogens reduced the overall plant growth and biomass (Table 3). The bioagent inoculated plants had higher biomass of 0.51-0.60 and 0.98-1.15 g plant-1 on 90 and 180th days growth, respectively, in comparison to 0.46 and 0.84 g plant-1 in uninoculated control. Maximum gain was observed in case of P. tinctorius followed by L. laccata. After 90 days growth, the use of biocontrol agents individually, significantly improved seedling height (5.9-6.5 cm) over uninoculated control (4.8 cm), with maximum gain in blue pine seedlings inoculated with P. tinctorius, followed by L. laccata, T. viride and T. harzianum. With the advancement in growth period, the shoot height depicted almost similar trend with inoculated plants having height of 9.2-10.6 cm as compared to 7.5 cm in unioculated control after 180 days growth.
Table 3: |
In vivo interaction of fungal antagonists with wilt
pathogens, Rhizoctonia solani (R) and Fusarium oxysporum
(F) on blue pine seedlings (pooled data of 2 years) |
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* Root rot scale: No root rot = 0; <10 percent root area affected = 0.10; 11-25 percent root area affected = 0.25; 26-50 percent root area affected = 0.50; 51-75 percent root area affected = 0.75; 76 percent root area affected = 1.00 |
Rhizoctonia-infection resulted in less plant biomass (0.31 g plant-1)
and seedling height (3.1 cm) after 90 days growth, with almost similar trend
noticed after 180 days. However, in presence of bioagents the effect of R.
solani was significantly reduced and seedling biomass and shoot height in
pine was 0.39-0.46 g plant-1 and 3.5-4.1 cm, respectively, on 90th
day of growth. The seedlings biomass and height was 0.69-0.80 g plant-1
and 5.7-6.6 cm, respectively, in Rhizoctonia + bioagent treatments as
compared to 0.56 g plant-1 and 5.0 cm in R. solani treatment
alone on 180th day of growth with maximum increase in R. solani + P. tinctorius
followed by R. solani + L. laccata treatment. The bioagents
in presence of root rot pathogen Fusarium oxysporum also promoted plant
growth and mitigated the influence of pathogen (Table 3).
Fusarium oxysporum infected blue pine seedlings had less biomass and
shoot height of 0.22 g plant-1 and 2.4 cm, respectively, after 90
days growth and 0.41 g plant-1 and 4.1 cm after 180th days growth
in comparison to unicoculated control. The bioagents in F. oxysporum-infected
soils remarkably had higher plant biomass and seedling height ranging from 0.28
to 0.32 g plant-1 and 2.6 to 3.2 cm, respectively, on 90th day of
growth and from 0.52-0.60 g plant-1 and 4.8-5.4 cm, respectively,
on 180th day of growth with maximum improvement in biomass in F. oxysporum+T.
harzianum, followed by F. oxysporum+P. tinctorius and maximum
increase in plant height in F. oxysporum+T. viridi treatment followed
by F. oxysporum+T. harzianum. The overall improvement in plant
biomass and seedling height by bioagents may be attributed to the growth promoting
and protective effects of biocontrol agents. Werner et
al. (2002) observed that mycorrhizal Pinus sylvestris seedlings
inoculated with Trichoderma virens produced significantly higher plant
growth and biomass of needles, trunks and root than uninoculated plants.
T. harzianum showed stimulatory effect on seedling growth and biomass of
pine. Trichoderma species reportedly produce hormone like metabolites
and release nutrients from soil or organic matter thereby facilitate better
plant growth (Windham et al., 1986; Yobo
et al. 2010).
Disease control by antagonists: The antagonists remarkably decreased
root rot disease index and improved root colour intensity in pathogen-infected
treatments (Table 3). High root rot index was observed in
pine seedlings raised in either R. solani or F. oxysporum infested
soil as compared to control (non-sick soil). The bioagents significantly reduced
root rot from 38.3% in R solani treatment to 22.6-26.7% with maximum
decrease in L. laccata followed by P. tinctorius, T. harzianum
and T. viridi. In case of F. oxysporum infection, the bioagents
decreased root rot from 47.2% in F. oxysporum treatment to 29.4-35.4%
in bioagents, with maximum reduction by L. laccata, followed by P.
tinctorius, T. harzianum and T. viridi. The study revealed that,
besides improving growth, bioagents especially mycorrhiza protected seedlings
against root rot pathogens in blue pine. Inoculation of bioagents in presence
of root rot pathogens proved superior over pathogen-inoculated control. Decrease
in root rot disease index by antagonistic fungi including mycorrhiza may be
attributed to the competition for nutrient base and space, cross protection
of roots, alteration in host metabolism and improved nutrient supply. Chakravarty
and Adam (1987) reported that growth and development of mycorrhizal species
was strongly suppressed by root rot pathogens F. oxysporum and R.
solani. Various bioagents reportedly have greater rhizosphere competence
and parasitizes the pathogenic fungi (Naik, 2003; Gao
et al., 2010). The antagonists in rhizosphere likely compete with
the pathogen for host surface and nutrients and inhibit pathogenic growth through
antibiosis/mycoparasitism mechanisms (Howell, 2003;
Sharma et al., 2010). This seems to have reduced
the seedling root decay. These speculations are substantiated by our in vitro
bicontrol studies on F. oxysporum and R. solani. The principal
mechanisms of Trichoderma spp. for disease control have been presumed
to be those primarily acting upon the pathogens and include mycoparasitism,
antibiosis and competition for resources and space (Harman,
2006). Thus reduction of root rot in blue pine seedlings incited by F.
oxysporum and R. solani may be due to the biological control action
of T. harzianum on these pathogens.
Pisolithus tinctorius alone showed 44.2% root colonization in comparison
to 39.1% root colonization observed in L. laccatta after 180 days growth.
In presence of R. solani the mycorrhizal root colonization was comparatively
less, 24.2% in P. tinctorius and 19.5% in L. laccata. Almost similar
effect of mycorrhiza was noticed in presence of F. oxysporum. Increased
root colonization may be attributed to the competition for space and reduction
in root biomass due to root decay by rot causing incitant, thereby leaving less
surviving root area for symbiosis. The seedlings with considerable ectomycorrhizal
colonization rapidly regenerate new lateral roots, create more new sites for
ectomycorrhizae and thereby utilize available nutrients more efficiently than
non-mycorrhizal seedlings. Antagonists or mycorrhizae inoculation in Rhizoctonia
infected plants lessened the effect of root rot. Mycorrhizal root colonization
was less in presence of Rhizoctonia solani and F. oxysporum. This
indicates that the disease protection by ectomycorrhiza may involve multiple
mechanisms including antibiosis, synthesis of fungistatic compounds by plant
roots in response to mycorrhizal infection and physical barrier of fungal mantle
around the plant root (Duchesne et al., 1987;
Dar et al., 2007). The favourable effect of mycorrhizal
fungus on plant growth and health may be attributed to the excretion of growth
promoting substances by mycorrhizae or indirectly by alteration in root physiology,
uptake of minerals and pattern of exudation into the mycorrhizosphere (Leyval
and Berthelin, 1990).
CONCLUSION
The tested fungus biocontrol agents, including mycorrhiza, effectively mitigated
root rot disease in blue pine. The bioagents once applied in field multiply
in soil and when threshold population is achieved they may reduce the disease
incidence thereby ensure the successful establishment of pine seedlings at early
stages. The fungal bioagents can be efficiently exploited in integrated disease
management module.
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