The coniferous forests of Kashmir Himalayas provide a sustainable habitat for wide varieties of ectomycorrhizal fungi. The identification and characterization of many of these fungi however largely involves morphological descriptions of sporocarps alone, thus sometimes raising questions about the authenticity of these studies. The present study was carried out to identify and characterize ectomycorrhizal fungi from the coniferous forests of Kashmir Himalaya using both morphological and molecular methods. Herein we report on the identification and characterization of three potential ectomycorrhizal
Ectomycorrhizas are mutualistic symbiotic associations formed between roots of higher plants with certain soil fungi. The mycorrhizal fungi benefit associated host trees in a number of ways although the most important is by enhancing nutrient mobilization particularly for elements with low mobility in the soil (N and P) and several micronutrients, release of nutrients from mineral particles or rock surfaces via weathering, effects on carbon cycling, interactions with mycoheterotrophic plants, mediation of plant responses to stress factors such as drought, soil acidification, toxic metals, and plant pathogens, and rehabilitation and regeneration of degraded forest ecosystems, as well as a range of possible interactions with groups of other soil microorganisms [
The Jammu and Kashmir state located in the far north of India is a mountainous area in the northwest Himalayas that shares international boundaries with Pakistan in the west and China in the northeast. Punjab and Himachal Pradesh are its neighbouring states within the country. The state has a geographic area of 101387 sq. km. It lies between 32°17′ and 37°05′ north latitude and 72°31′ and 80°20′ east longitude. The state is divided into three geographic regions: Ladakh, Kashmir Valley, and Jammu. The Kashmir Valley lies between 33°20′ and 34°54′ N latitude and 73°55′ and 75°35′ E longitudes covering an area of 15,948 sq. km and harbors diverse coniferous forests. The coniferous forests of Kashmir valley support diverse populations of
Together with classical taxonomical methods, molecular methods may be useful and helpful for the correct identification of mushroom species. Through molecular methods most of the fungi have been identified by comparative analyses of the ribosomal DNA sequences, especially the ITS region. For example, Peintner et al. [
In this study, the combined morphological and molecular nucleotide analysis of various
The ectomycorrhizal sporocarps were collected from different coniferous forest areas of Kashmir Himalaya, like Gulmarg, Kokernag, Daksum, Drang, Mammer, and so forth. The colour of the sporocarps was recorded at the time of collection. Colour of sporocarps was described based on the codes of Kornerup and Wanscher (1978). Standard methods were followed for the collection of sporocarps [
The molecular characterization of sporocarps involved sequencing of internal transcribed spacer (ITS) region of the nuclear ribosomal genes (rDNA). For this, genomic DNA was isolated from sporocarps and roots of collected species.
Genomic DNA was isolated from fresh sporocarps by CTAB method. For this 200–250 mg of material was taken and grinded into fine powder with the aid of liquid nitrogen. The powder was then taken in 15 mL centrifuge tube and to this 5 mL prewarmed CTAB buffer (1 M Tris HCl pH 8.0, 5 M NaCl, 0.5 M EDTA pH 8.0, CTAB, 2%
The ITS region of rDNA was amplified by polymerase chain reaction (PCR) with ITS1 and ITS4 primers in Applied Biosystems 2720 Thermal Cycler. The amplified fragment includes ITS1, 5.8S, and the ITS2 of rDNA. The 50
The sequenced PCR amplicons were BLAST (Basic Local Alignment Search Tool) searched using the National Center for Biotechnology Information (NCBI), USA, database for comparison of sequences. The initial alignment of all sequences was directly made with the ClustalX multiple alignment program [
For phylogenetic analysis closely related sequences were retrieved from GenBank. The sequence alignments were performed using Molecular Evolutionary Genetics Analysis (MEGA) software [
Three ectomycorrhizal species from genus
Pileus (Pers. ex Fr.): cap 2–5 cm broad, shape convex or conical when young, then expanded and umbonate with age, surface subviscid, silky, dark brown when moist, especially at the centre, drying pale fawn, covered with minute white fibrous scales; flesh pale orange coloured, no colour change on bruising. Lamellae: gills close and crowded; attachment adnate; dark brown often with violet tinge. Stipe: stem 3–8 cm long, 1-2 cm thick at the base, clavate; solid and firm when young, viscid; brownish, covered at first with the white cottony veil which forms a distinct but short-lived ring and cottony scales below. Spores: spores broadly elliptic, spore print rust brown. Habit: naucorioid; growth type: solitary occasionally scattered in coniferous forests.
Field photograps of
Pileus: cap 5–10 cm broad; convex or conical when young, with age becomes broadly convex; surface sub viscid, silky, smooth with appressed fibrils over the entire surface, fibrils more towards margin; colour light yellow to orange, with age colour faints; margin incurved, entire, not splitting at maturity; flesh pale orange coloured, no colour change on bruising. Lamellae: gills attached with stem, attachment adnate, space moderate, coloured like the cap, covered with orange coloured cortina when young. Stipe: 2–8 cm long, 1-2 cm thick; attachment central; more or less equal; dry; silky; pale orangish above, cream white colored below; fibrillar orange coloured cortina at stipe apex; flesh pale orangish; odor: mild. Habit: naucorioid; growth type: solitary occasionally scattered.
Pileus: cap 5–10 cm broad, shape convex, with age becomes broadly convex to flat, or sometimes bell-shaped; surface moist and sticky; margin entire, incurved, splitting at maturity; colour generally grayish when young, with age becomes brown with deep dark brown shades; flesh white, no colour change on bruising or on exposure to air. Lamellae: gills attached with stem, attachment adnate; close; colour light brow to gray, with age becoming rusty brown. Stipe: stem 5–8 cm long, 2-3 cm thick; club shaped; surface dry; colour whitish, with age discolouring brownish, occasionally purplish scales preset at apex when young; apex remains adhering with rusty cortina remnants; flesh white and firm. Spores: spore print rusty brown. Habit: naucorioid; growth type: solitary occasionally scattered.
The molecular characterization was performed by carrying out sequencing of rDNA ITS region. The ITS region amplified with ITS1 and ITS4 pair of primers varied in length from 650 to 750 bp in the three species (Figure
GenBank accession numbers and top BLAST match sequences of the mushroom isolates along with maximum identity, query coverage.
Accession number | BLAST match sequence | ||
---|---|---|---|
Reference accession number | Coverage | Maximum identity | |
KC859462 | AY669683.1 |
90 | 92 |
AF430262.1 |
89 | 90 | |
JF907921.1 |
88 | 90 | |
FJ827156.1 |
89 | 90 | |
GQ159816.1 |
86 | 89 | |
FN428982.1 |
85 | 89 | |
AF389156.1 |
84 | 85 | |
EU259284.1 |
84 | 89 | |
FJ039542.1 |
80 | 85 | |
HQ604727.1 |
80 | 84 | |
|
|||
KF023073.1 | AY669677.1 |
100 | 99 |
FJ039534.1 |
99 | 97 | |
HQ604701.1 |
100 | 97 | |
HQ604714.1 |
100 | 97 | |
FJ039552.1 |
100 | 97 | |
FJ039562.1 |
100 | 97 | |
HQ604706.1 |
100 | 97 | |
FJ039551.1 |
100 | 97 | |
AY669672.1 |
100 | 97 | |
AY669658.1 |
100 | 97 | |
|
|||
KF727563 | FJ039612.1 |
100 | 99 |
HQ604687.1 |
100 | 98 | |
HQ604688.1 |
100 | 98 | |
AY669536.1 |
96 | 97 | |
FJ717596.1 |
99 | 94 | |
HQ604653.1 |
99 | 94 | |
FJ717595.1 |
99 | 94 | |
DQ384593.1 |
99 | 94 | |
FJ039602.1 |
99 | 94 | |
FJ039601.1 |
99 | 94 |
1% agarose gel showing amplified ITS rDNA PCR products of 3 different ECM
The identity of species was further confirmed by performing phylogenetic analysis of the species in neighbour-joining method. The closely related top matched BLAST sequences with which the present study isolates showed maximum identity were retrieved from GenBank for phylogenetic analysis with present study species accessions. The phylogenetic analysis of the large dataset including 38 ITS sequences of
(a) Pairwise nucleotide divergence among the various accessions with which KC859462.1 showed maximum similarity. (b) Pairwise nucleotide divergence among the various accessions with which KF023073.1 showed maximum similarity. (c) Pairwise nucleotide divergence among the various accessions with which KF727563.1 showed maximum similarity.
KC859462 | |||||||||||
AY669683.1 |
0.000 | ||||||||||
AF430262.1 |
0.003 | 0.032 | |||||||||
JF907921.1 |
0.004 | 0.024 | 0.026 | ||||||||
HM240523.1 |
0.004 | 0.030 | 0.030 | 0.020 | |||||||
GQ159816.1 |
0.034 | 0.030 | 0.030 | 0.020 | 0.000 | ||||||
FN428982.1 |
0.034 | 0.034 | 0.034 | 0.030 | 0.026 | 0.026 | |||||
AF389156.1 |
0.034 | 0.030 | 0.030 | 0.020 | 0.000 | 0.000 | 0.026 | ||||
EU821693.1 |
0.034 | 0.030 | 0.030 | 0.020 | 0.000 | 0.000 | 0.026 | 0.000 | |||
FJ039542.1 |
0.037 | 0.030 | 0.005 | 0.024 | 0.028 | 0.028 | 0.032 | 0.028 | 0.028 | ||
FN428979.1 |
0.032 | 0.024 | 0.026 | 0.000 | 0.020 | 0.020 | 0.030 | 0.020 | 0.020 | 0.024 | |
HQ604726.1 |
0.395 | 0.395 | 0.392 | 0.384 | 0.389 | 0.389 | 0.391 | 0.389 | 0.389 | 0.398 | 0.384 |
KF023073 | ||||||||||
AY669677.1 |
0.001 | |||||||||
FJ039534.1 |
0.009 | 0.032 | ||||||||
HQ604701.1 |
0.032 | 0.024 | 0.026 | |||||||
HQ604714.1 |
0.034 | 0.030 | 0.030 | 0.020 | ||||||
FJ039552.1 |
0.034 | 0.030 | 0.030 | 0.020 | 0.000 | |||||
FJ039562.1 |
0.034 | 0.034 | 0.034 | 0.030 | 0.026 | 0.026 | ||||
HQ604706.1 |
0.034 | 0.030 | 0.030 | 0.020 | 0.000 | 0.000 | 0.026 | |||
FJ039551.1 |
0.034 | 0.030 | 0.030 | 0.020 | 0.000 | 0.000 | 0.026 | 0.000 | ||
AY669672.1 |
0.037 | 0.030 | 0.005 | 0.024 | 0.028 | 0.028 | 0.032 | 0.028 | 0.028 | |
AY669658.1 |
0.032 | 0.024 | 0.026 | 0.000 | 0.020 | 0.020 | 0.030 | 0.020 | 0.020 | 0.024 |
KF727563 | ||||||||||
FJ039612.1 |
0.001 | |||||||||
HQ604687.1 |
0.004 | 0.004 | ||||||||
HQ604688.1 |
0.004 | 0.004 | 0.000 | |||||||
AY669536.1 |
0.011 | 0.011 | 0.012 | 0.012 | ||||||
FJ717596.1 |
0.049 | 0.044 | 0.044 | 0.044 | 0.041 | |||||
HQ604653.1 |
0.044 | 0.041 | 0.040 | 0.040 | 0.038 | 0.037 | ||||
FJ717595.1 |
0.047 | 0.043 | 0.042 | 0.042 | 0.039 | 0.016 | 0.034 | |||
DQ384593.1 |
0.044 | 0.040 | 0.039 | 0.039 | 0.037 | 0.039 | 0.006 | 0.034 | ||
FJ039602.1 |
0.045 | 0.043 | 0.042 | 0.042 | 0.040 | 0.036 | 0.035 | 0.035 | 0.036 | |
FJ039601.1 |
0.046 | 0.041 | 0.041 | 0.041 | 0.041 | 0.035 | 0.020 | 0.031 | 0.021 | 0.034 |
Phylogenetic relationship of present study ITS sequences ▲ with other related members based on maximum likelihood method inferred from ITS sequences.
The coniferous forests of Kashmir Himalaya support diverse ectomycorrhizal communities due to varied and diverse climatic conditions. The ECM species richness of the region is directly related to its diverse forest communities and weather patterns, but all the regions of the state have not been extensively surveyed and explored for ectomycorrhizal fungi fruiting in different seasons and associated with different host trees till now. Most of the species reported from Kashmir Himalaya have been identified solely on the basis of morphological and microscopic characters. Morphological identification of ECM sporocarps is difficult and requires profound knowledge and experience; it is prone to mistakes due to the frequent homoplasy of phenetic characters. In addition phenotypic variation in fungi can be affected by substrate and environmental factors limiting the application of morphological characters in the identification of ECM sporocarps [
The combined approach of morphological and molecular biology lets us identify and characterize the field collected ectomycorrhizal sporocarps of three
The species of
Analysis of basidiocarps using ITS primers (ITS1 and ITS4 and their derivatives) and PCR analysis had proven very useful and easy method for identifying particular ECM species [
The combined approach of morphological and molecular analysis can enrich and provide additional information to mushroom biodiversity and GenBank database and can also lead to the discovery of many new unidentified species from the region.
The authors declare no financial or nonfinancial conflict of interests.
The authors greatly appreciate Senior and Junior Research Fellowships in favour of Sheikh Tahir Majeed (09/251/0055/2013/EMRI) and Qussin Basharat (09/251/0045/2011/EMRI) from Council of Scientific and Industrial Research (CSIR), respectively. Special thanks are due to Sheetal Ambardar (School of Biotechnology University of Jammu) for her help in developing the phylogenetic analysis and providing valuable comments.