Alkaliphilic and thermostable lipase production by leaf litter fungus Leptosphaerulina trifolii A SMR-2011.

Fungi that inhabit fire-prone forests have to be adapted to harsh conditions and fungi affiliated to Ascomycota recovered from foliar litter samples were used for bioprospecting of molecules such as enzymes. Agni's fungi isolated from leaf litter, whose spores are capable of tolerating 110 oC were screened for thermostable lipases. One of the isolates, Leptosphaerulina trifolii A SMR-2011 exhibited high positive lipase activity than other isolates while screening through agar plate assay using Tween 20 in the medium. Maximum lipase activity (173.2 U/mg) of L. trifolii was observed at six days of inoculation and decreased thereafter. Among different oils used, the maximum lipase activity was attained by soybean oil (940.1 U/mg) followed by sunflower oil (917.1 U/mg), and then by mustard oil (884.8 U/mg), showing its specificity towards unsaturated fatty acids. Among the various organic nitrogen sources tested, soybean meal showed maximum lipase activity (985.4 U/mg). The partially purified enzyme was active over a wide range of pH from 8 to 12 with a pH optimum of 11.0 (728.1 U/mg) and a temperature range of 60-80 oC with an optimal temperature of 70 oC (779.1 U/mg). The results showed that lipase produced by L. trifolii is alkali stable and retained 85% of its activity at pH 11.0. This enzyme also showed high thermal stability retaining more than 50% of activity when incubated at 60 oC to 90 °C for 2 h. The ions Ca2+ and Mn2+ induced the lipase activity, while Cu2+ and Zn2+ ions lowered the activity compared to control. These results suggests that the leaf litter fungus L. trifolii serves as a potential source for the production of alkali-tolerant and thermostable lipase.

Isolation and characterization of thermostable and alkali-tolerant cellulase from litter endophytic fungus Bartalinia pondoensis.

Endophytic fungi in plant tissues produce a wide range of secondary metabolites and enzymes, which exhibit a variety of biological activities. In the present study, litter endophytic fungi were isolated from a fire-prone forest and screened for thermostable cellulases. Among nine endophytic fungi tested, two isolates, Bartalinia pondoensis and Phoma sp., showed the maximum cellulase activity. Bartalinia pondoensis was further selected for its cellulase production and characterization. Among the carbon and nitrogen sources tested, maximum cellulase production was observed with maltose and yeast extract, and the eucalyptus leaves and rice bran served as the best natural substrates. The cellulase activity increased with increasing temperature, with maximum activity recorded at 100 °C. The maximum CMCase activity was observed between pH 6.0 and 7.0 and retained 80% of its activity in the pH range of 8-10. Partially purified cellulase of B. pondoensis retained 50% of its activity after 2 h of incubation at 60 °C, 80 °C and 100 °C. These results suggest that litter endophytic fungus B. pondoensis is a potential source for the production of thermostable and alkali-tolerant cellulase.

Seed Treatment With Systemic Fungicides: Time for Review.

Pre-sowing seed treatment with systemic fungicides is a firmly entrenched practice for most agricultural crops worldwide. The treatment is intended to protect the crop against seed- and soil-borne diseases. In recent years, there is increasing evidence that fungicidal applications to manage diseases might inadvertently also affect non-target organisms, such as endophytes. Endophytes are ubiquitously present in plants and contribute to plant growth and development besides offering resistance to biotic and abiotic stresses. In seeds, endophytes may play a role in seed development, seed germination, seedling establishment and crop performance. In this paper, we review the recent literature on non-target effects of fungicidal applications on endophytic fungal community and discuss the possible consequences of indiscriminate seed treatment with systemic fungicide on seed endophytes. It is now well recognized that endophytes are ubiquitously present in all parts of the plant, including the seeds. They may be transmitted vertically from seed to seed as in many grasses and/or acquired horizontally from the soil and the environment. Though the origins and evolution of these organisms in plants are a matter of conjecture, numerous studies have shown that they symbiotically aid in plant growth and development, in nutrient acquisition as well in protecting the plants from abiotic and biotic stresses. Against this background, it is reasonable to assume that the use of systemic fungicides in seed treatment may not only affect the seed endophytes but also their attendant benefits to seedling growth and establishment. While there is evidence to indicate that fungicidal applications to manage plant diseases also affect foliar endophytes, there are only few studies that have documented the effect of seed treatment on seed-borne endophytes. Some of the convincing examples of the latter come from studies on the effect of fungicide application on rye grass seed endophyte AR37. More recently, experiments have shown that removal of seed endophytes by treatment with systemic fungicides leads to significant loss of seedling vigour and that such losses could be partially restored by enriching the seedlings with the lost endophytes. Put together, these studies reinforce the importance of seed endophytes to seedling growth and establishment and draw attention on how to trade the balance between the benefits of seed treatments and the direct and indirect costs incurred due to loss of endophytes. Among several approaches, use of reduced-risk fungicides and identifying fungicide-resistant endophytes are suggested to sustain the endophyte contribution to early seedling growth.

Fungal Endophyte-Mediated Crop Improvement: The Way Ahead.

Endophytes are non-disease causing microbes (bacteria and fungi) surviving in living tissues of plants. Their intimate association and possible coevolution with their plant partners have resulted in them contributing to an array of plant growth benefits ranging from enhanced growth and biomass accumulation, tolerance to abiotic and biotic stresses and in nutrient acquisition. The last couple of decades have witnessed a burgeoning literature on the role of endophytes (Class 3 type) in regulating plant growth and development and their adaptation to abiotic and biotic stresses. Though the underlying mechanisms of plant-endophyte interactions are far from clear, several studies have raised the hope of their potential application in agriculture, especially in mitigating abiotic and biotic stresses. The use of endophytes is envisaged as a route to reduce the production cost and burden on the environment by lessening the dependence on breeding for crop improvement and agrochemicals. Unfortunately, save a few well documented examples of their use, a little of these insights has been translated into actual agricultural applications. Here, we reflect on this paucity and elaborate on some of the important bottlenecks that might stand in way of fully realizing the potential that endophytes hold for crop improvement. We stress the need to study various facets of the endophyte-plant association for their gainful application in agriculture.

Characterization of an ionic liquid-tolerant ß-xylosidase from a marine-derived fungal endophyte.

Ionic liquids (ILs) are used in lignocellulosic biomass (LCB) pretreatment because of their ability to disrupt the extensive hydrogen-bonding network in cellulose and hemicellulose, and thereby decrease LCB recalcitrance to subsequent enzymatic degradation. However, this approach necessitates the development of cellulases and hemicellulases that can tolerate ∼20% (w/v) IL, an amount that either co-precipitates with the sugar polymers after the initial pretreatment or is typically used in single-pot biomass deconstructions. By investigating the secretomes from 4 marine-derived fungal endophytes, we identified a ß-xylosidase derived from Trichoderma harzianum as the most promising in terms of tolerating 1-ethyl-3-methylimidazolium-dimethyl phosphate (EMIM-DMP), an IL. When tested with p-nitrophenyl-ß-d-xyloside, this extracellular xylosidase retained ∼50% activity even in 1.2 mol·L-1 (20% w/v) EMIM-DMP after incubation for 48 h. When tested on the natural substrate xylobiose, there was ∼85% of the initial activity in 1.2 mol·L-1 EMIM-DMP after incubation for 9 h and ∼80% after incubation for 48 h. Despite previous findings associating thermostability and IL tolerance, our findings related to the mesophilic T. harzianum ß-xylosidase(s) emphasize the need to include the marine habitat in the bioprospecting dragnet for identification of new IL-tolerant LCB-degrading enzymes.

Endolichenic fungi: the lesser known fungal associates of lichens.

Lichens are the result of a stable mutualism between a fungal and a photosynthesising partner (alga or cyanobacterium). In addition to the fungal partner in this mutualism, lichens are associated with endolichenic fungi which reside inside their thalli. The endolichenic fungi appear to have evolved with the lichen and many of them are a source of novel metabolites vested with unique bioactivities. There is very little information on the biology of endolichenic fungi and their interactions with the other components of a lichen microbiome. There is an urgent need to understand these aspects of endolichenic fungi such that their ecology and economic potential are known more completely. The current knowledge on endolichenic fungi is reviewed here.

Incidence of Leptosphaerulina crassiasca in symptomless leaves of peanut in southern India.

A Loculoascomycete fungus belonging to the genus Leptosphaerulina was found to survive as symptomless endophyte in the leaflets and rachis of peanut plant (TMV 7). Based on morphological and colony characteristics, it is identified as Leptosphaerulina crassiasca, a fungus that causes the pepper spot and leaf scorch diseases in peanut. Although several common endophytic fungi were recovered from the leaves of peanut, L . crassiasca showed a high relative density of infection. Owing to its endophytic nature, its pathogenicity could not be proved by infection studies. However, its identification as L. crassiasca suggests that this pathogen survives as symptomless endophyte in its host. There are limited studies on fungal endophytes of cultivated crops and hence, the present observation underlines the need to know more about the biology of endophytic fungi of crop plants.

Endophytic fungi associated with cacti in Arizona.

21 cactus species occurring in various localities within Arizona were screened for the presence of fungal endophytes. 900 endophyte isolates belonging to 22 fungal species were isolated. Cylindropuntia fulgida had the maximum endophyte species diversity, while C. ramosissima harboured the maximum number of endophyte isolates. Alternaria sp., Aureobasidium pullulans, and Phoma spp. were isolated from several cactus species. The diversity of the endophyte assemblages was low and no host specificity among endophytes was observed. However, the frequencies of colonization of the few endophyte species recovered were high and comparable to those reported for tropical plant hosts. Species of Colletotrichum, Phomopsis, and Phyllosticta, which are commonly isolated as endophytes from plants of more mesic habitats, were absent from these cacti.

Characterization of the melanin pigment of a cosmopolitan fungal endophyte.

Phyllosticta capitalensis (teleomorph Guignardia mangiferae) occurs as a foliar endophyte in woody trees belonging to different families of both temperate and tropical regions. We isolated this endophyte from plants in different habitats, such as mangroves, dry deciduous forest, moist deciduous forest and semi-evergreen forest. This endophyte was found to produce a black pigment that was characterized to be melanin based on uv-visible, IR and ESR spectra and chemical tests. Tricyclazole, a specific inhibitor of pentaketide melanin biosynthesis, inhibited synthesis of the pigment indicating it is a 1-8, dihydroxynaphthalene. This appears to be the first report of such a melanin in Phyllosticta or other foliar endophytes. Melanin in the hyphae of P. capitalensis may be responsible for the success of this fungus as a cosmopolitan endophyte, since melanin is known to enhance the survival capability of fungi in stressful environments.

ITS-RFLP and ITS sequence analysis of a foliar endophytic Phyllosticta from different tropical trees.

Different isolates of a foliar endophytic species of Phyllosticta were isolated from different tropical tree species in India to examine genetic variation among the isolates. Internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis did not detect any variation among the isolates, suggesting that they all belong to the same species. Sequence data of the ITS region of ribosomal DNA (ITS1 and ITS2, including 5.8S rDNA) supported the identity of the present fungal isolates as P. capitalensis. These results show that P. capitalensis (teleomorph Guignardia endophyllicola?) is an ubiquitous foliar endophyte that can infect tree hosts from different families and habitats.