Characterization of arbuscular mycorrhizal fungi in apple ( Malus domestica Borkh ) growing area in Kashmir Himalaya ( India ) : A case study of Bandipora district

Rhizosphere soil samples and root pieces of 3 year old apple trees (var. Red delicious) were collected from 10 villages of district Bandipora of Kashmir Himalaya. Samples were processed for isolation of arbuscular mycorrhizal spores and their identification. On the basis of various morphological characters such as spore shape, colour, size, hyphal colour and with the help of International Collection of Vesicular-Arbuscular Mycorrhizal (INVAM), the isolated genera were identified as Acaulospora, Scutellospora, Gigaspora, and Glomus. Glomus spores were more predominant in the district followed by Acaulospora. Spores of Gigaspora were larger in diameter while as Glomus spores were smaller. Glomus species showed higher root colonization from Markondal followed by Acaulospora species from Gorura, Gigaspora species from Ajas and Scutellospora species from Sangri. The highest biological activity was due to adequate application of organic manures in the soil and also due to application of fertilizers.


INTRODUCTION
Apple (Malus domestica Borkh) is considered as one of the most important and widely grown fruits in temperate zones of the world with regard to its acreage, production, economic returns and high nutritive value.Apple is the fourth widely produced fruit in the world after banana, orange and grapes.Indian apple production averages nearly 1.4 million making it the sixth largest apple producer in the world (Satish et al., 2006) and second in Asia (Deepa, 2008).In India, apple is mainly grown in three mountainous states of North India, viz.Himachal Pradesh, Jammu and Kashmir and Uttarakhand where they are typically grown at an altitude of 4000 to 11000 ft.*Corresponding author.E-mail: misbamalik98@gmail.com.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Jammu and Kashmir and Himachal Pradesh have almost equal area under apple, but later has the highest average yield and accounts for 67% of the total apple production (Masoodi, 2003) and 50% of its export in the country, earning a substantial foreign exchange.Productivity is much higher than national average of 6.86 tons/ha.India annually exports apple worth Rs.400 million (Nearly US $ 10 million) out of which apples worth Rs.200 million come from Kashmir.Moreover, it provides job opportunity to 1.2 million people directly or indirectly.In horticulture sector, the largest area of 43.53% is occupied by apple out of total area under fruit and 65.46% out of fresh fruit area (Anonymous, 2007), thereby making it the largest contributor to the state GDP among the horticulture produce.
Bandipora is a newly carved district from erstwhile Baramulla district.The district is surrounded by Himalayan Mountains having district Kargil on north, Kupwara in west, Baramulla in south and Ganderbal in east.The district is situated between 34°25´ and 34°42´ N Latitude and 74°39´ and 74°65´ E longitude.It has unique agro climatic conditions of low temperature even during summer months.The main fruit crop of the district is apple.Out of the total area of 140156 hactare under apple in Kashmir district Bandipora covers area of 5605 hactare (Anonymous, 2013).Bandipora is known for the better quality apple in the valley.
Phosphorus which is the second primary nutrient after nitrogen has a direct effect on yield and tree health.It is also important in determining fruit size, firmness, colour and storage potential.It increases the potential of fruiting by increasing the number of flower clusters, their intensity and the level of fruit set.But, phosphorus is an extremely immobile element in soils and even if it was added to soil in soluble form, it becomes immobilized as organic phosphorus, calcium phosphates, or other fixed forms (Jackson and Mason, 1984).AM-fungi are known to be effective in increasing phosphorus uptake in many crops, particularly in low phosphorus soils (Osonubi et al., 1991).They are associated with improved growth of many plant species due to increased nutrient uptake, production of growth promoting substances, tolerance to drought and synergistic interaction with other beneficial soil micro-organisms such as N-fixers and P-solubilizers (Sreenivasa and Bagyaraj, 1989).Arbuscular mycorrhizal fungi play an important role in sustainable agriculture as well as agricultural ecosystem management.The important genera of endomycorrhizal fungi reported so far are Acaulospora, Entrophosphora, Gigaspora, Glomus, Sclerocystis and Scutellospora (Morton, 1988).All these fungi are obligately associated with plant roots and develop symbiotic relationship with their hosts.Different species can be differentiated on the basis of sporocarp size, spore dimension, presence/absence of hyphal mantles, spore ornamentation (warts, wrinkles, pits, reticulum, spines, etc.), spore walls, spore content, hyphal attachment, manner of spore germination, histochemical reaction, etc. (Schenck and Smith, 1982).Keeping in view the importance of arbuscular mycorrhizal fungi in phosphorus solubilization and very little work having been done on arbuscular mycorrhizae with respect to apple, the present investigation was taken up with the objective of isolation and purification of arbuscular mycorrhizal spores from the rhizospheric soil of apple, morphological characterization of the spores upto generic level and their root colonization studies.

Study area
Bandipora is situated on the banks of the Wular, the largest freshwater lake in Asia, with geographic coordinates of 34.5052° N, 74.6869° E. In Bandipora district, apple production has swelled to 69,147 MT in 2014-15 from 65,102 MT in 2013-14.Similarly, the area under crop extended from 5,605 to 5,840 hectares in one year and thus contributes a lot in horticulture sector of Jammu and Kashmir.Ten villages (Arigam, Ajas, Asham, Gorura, Nadihal, Naidkhai, Sumbal, Markondal, Sangri, Watlab) were selected from district Bandipora.From each village three orchards were randomly chosen and from each orchard five rhizosphere soil samples were drawn which were composited into one representative sample.Most of the orchardists followed the pesticide schedule as per Department of Horticulture, Government of Jammu and Kashmir (Anonymous, 2015).

Isolation and purification of arbuscular mycorrhizal spores
Rhizosphere soil samples were collected from the feeder roots on all sides of the canopy of the tree (fruiting stage).The soil samples taken from the rhizosphere of apparently healthy apple trees were collected in June 2014.The soil samples (about 250 g each) were immediately brought to the laboratory, air-dried and processed for AM isolation using wet sieving and decantation method (Gerdemann and Nicolson, 1963).Counting of spores was done under microscope Olympus CH20i with magnification of 10×40.

Measurement of available sulphur, soil organic carbon, available phosphorus, available potassium
Available sulphur, soil organic carbon, available phosphorus, available potassium were studied as per Chesnin and Yein (1951), Walkley and Black (1934), Olsen et al. (1954), and Stanford and English (1949), respectively.Soil used during the present study contained 1.74% organic carbon, 357.43 kg/ha available nitrogen, 17.05 kg/ha phosphorus, 12.02 kg/ha available sulphur and 185.38 kg/ha available potassium and all these were in medium range.

Identification
The arbuscular mycorrhizal fungal species were morphologically identified on the basis of spore size, spore colour and spore wall up to the genera level as per guidelines by INVAM (http://invam.caf.wvu.edu).

Root colonization studies of the isolated spores
The isolated spores were further purified and mass multiplied on maize.Surface sterilized healthy maize seeds, pre-germinated in Petri plates under aseptic conditions, were sown in polythene bags containing sterilized soil + sand mixture (1:2 w/w).These bags were aseptically inoculated with identical AM spores at 5 cm depth (Jackson, 1973).The bags were kept in a greenhouse at 25±3°C and irrigated with sterile water.The plants were uprooted after 45 days.The roots were collected, washed with sterile water to remove adhering soil debris and observed for mycorrhizal infection.The infectivity was proved by noticing the presence of hartig net, vesicles, asbuscules or hyphae of endophytes on roots.
For estimating mycorrhizal root colonization, the root samples were collected and washed carefully to remove the adhering debris.The tertiary roots were cut into small pieces of approximately 1 cm length and subjected to differential staining as described by Phillips and Hayman (1970).The estimation of mycorrhizal infection in roots was made by visual observation (Giovannetti and Mosse, 1980).A randomly selected aliquot of stained root segments, suspended in water, was spread in a Petridish viewed under a dissecting microscope at a magnification of 10 and 40×.In case of AM colonization, root segments containing vesicles and arbuscules of endophyte and number of mycorrhizal short roots were considered infected as suggested by Beckjord et al. (1984).

Per cent mycorrhizal infection = Number of infected root segments / Total number of segments examined × 100
The data recorded during the investigation was statistically analyzed with the help of Pearson correlation (Gomez and Gomez, 1984).

Morphological characterization of arbuscular mycorrhizal spores
Spore morphology and wall characteristics were considered for the identification of arbuscular mycorrhizal fungi.The spores isolated from soils of district Bandipora were identified upto generic level using bibliographies provided by Walker et al. (2007).Four types of genera were isolated.The genera isolated were Acaulospora, Scutellospora, Gigaspora, and Glomus.There were 3 to 6 unidentified spores per gram from all studied locations (Figure 1).
Acaulospora spores were present singly in the soil and develop laterally on the neck of asporiferous saccule.Spores were light orange to yellowish brown (Table 1 and  Figures 4, 5, 6 and 7), globose to sub-globose in shape and 150 to 210 µm in diameter.These spores were triple layered with L1 which forms the spore surface light yellow to apricot yellow in colour and 0.7 to 2.0 µm in thickness.L2 was laminate and light orange to yellowish brown, 6.8 to 7.4 µm in thickness.L3 was laminate, hyaline, 0.8 to 1.6 µm in thickness and usually tightly adherent to L2.Similar observations have been reported by others also (Walker et al., 2007;Sharma et al., 2009).Gigaspora spore wall consisted of a permanent outer layer enclosing a laminate layer, each with different properties that distinguish species (e.g.color, thickness, etc).Our observations corroborate with those of Koske (1987) and Bentivenga and Morton (1995).Scutellospora spores were with or without ornamentations.Spores consisted of a bilayered spore wall and two bilayered flexible inner walls.Thin-walled auxillary cells with smooth to knobby surfaces were produced on hyphae in the soil near the root surface and were also reported by Schenck and Perez (1990).The spore colour of the species of Glomus was of wide range.It varied from red-brown to almost black or straw to dark orange but most were yellow brown in colour.Spores possessed globose to sub-globose shape, about 40 to 120 µm in size.Spore wall consisted of three layers (L1, L2 and L3).Our findings corroborate with those of many other workers (Koske, 1984;   Koske and Gemma, 1990).
There was no evidence of any ectomycorrhizal association with apple roots, and this corroborates with the findings of Greene et al. (1982).Glomus species was common and made up for more than 75% of total isolates followed by Acaulospora, Gigaspora and Scutellospora.Dominancy of Glomus in the present study is in agreement with the findings of many other workers (Mridha and Dhar, 2007;Burni et al., 2009;Sharma et al., 2009).The predominance of Glomus spp.under varying soil conditions might be due to the fact that they are widely adaptable to the varied soil conditions and survive in acidic as well as in alkaline soils (Pande and Tarafdar, 2004).

Root colonization studies of arbuscular mycorrhizal fungi
In the current study, the AM colonization in the apple roots from Bandipora district varied between 62.04 and 81.13% (Figure 2,3,8,9,10 and 11).The results are in conformity with the Kandula et al., (2006) who also observed higher colonization in the apple roots and confirmed the ubiquitous nature of AMF spores.The highest root colonization was recorded in response to the inoculation with Glomus spp.(81.13%) followed by Acaulospora species (75.34%),Gigaspora species (73.13%) and Scutellospora species (70.00%).Similar results were reported by some workers (Hosamani et al., 2004;Smith and Read, 2008).
Results of the present study indicate that the nutrient contents of the soils played a significant role in occurrence of different species of arbuscular mycorrhizal fungi and it is evident from the Perusal of the data presented in Table 2 which revealed that AM spore population of district Bandipora was positively and significantly correlated with organic carbon (r=0.752*).The results are in conformity with those of Lipinski et al. (2003) who also reported a significant positive correlation between soil organic carbon and AM spore population.Negative relationship of AM spore population with available phosphorus (-0.543*) content in the soil was also reported.Stribley et al. (1980) who also observed that infection developing under conditions of high phosphorus availability may function parasitically without making any beneficial contribution to plant nutrient supply.Our findings corroborate with those of Graham and Timmer (1984) and Wu et al. (2006).There was a significant correlation between AM spore population and root colonization (r=0.613*) in district Bandipora.Kumar et al. (2013) also found a significant positive correlation between mycorrhizal spores and colonization.Yang et al. (2010) found a positive correlation between and mycorrhizal colonization and spores.These results are also supported by Li et al. (2009).Since the climatic conditions of the study area fall under temperate zone which are conducive to the mycorrhizal development, it is possible that concentration of such propagules may be higher (Akhter, 2005).
Moreover, influence of apple roots through their exudates cannot be ruled out which needs further studies.highly put on record.

Figure 1 .
Figure 1. Isolation of arbuscular mycorrhizal spores from rhizospheric soil of apple from different locations of district Bandipora.

Figure 2 .
Figure 2. In-vitro root colonization by AM fungal spores isolated from district Bandipora.

Table 1 .
Morphological features of isolated genera of AM fungi.

Table 2 .
Correlation between spore population and other studied parameters of district Bandipora.
*Correlation is significant at the 0.05 level.