Effect of α-glycosidase inhibitors from endophytic fungus Alternaria destruens on survival and development of insect pest Spodoptera litura Fab. and fungal phytopathogens

In the present study the production of α-glycosidase inhibitors was used as a strategy to screen endophytic fungi with insecticidal and antifungal potential. Endophytic fungi were isolated from Calotropis gigantea L. (Gentianales: Apocynaceae) and evaluated for their α-glycosidase inhibitory activity. Maximum inhibitory activity was observed in an isolate AKL-3, identified to be Alternaria destruens E.G.Simmons on the basis of morphological and molecular analysis. Production of inhibitory metabolites was carried out on malt extract and partially purified using column chromatography. Insecticidal potential was examined on Spodoptera litura Fab. (Lepidoptera: Noctudiae). Partially purified α-glycosidase inhibitors induced high mortality, delayed the development period as well as affected the adult emergence and induced adult deformities. Nutritional analysis revealed the toxic and antifeedant effect of AKL-3 inhibitors on various food utilization parameters of S. litura. They also inhibited the in vivo digestive enzymes activity in S. litura. Partially purified α-glycosidase inhibitors were also studied for their antifungal potential. Inhibitors demonstrated antifungal activity against the tested phytopathogens inducing severe morphological changes in mycelium and spores. This is the first report on production of α-glycosidase inhibitors from A. destruens with insecticidal and antifungal activity. The study also highlights the importance of endophytes in providing protection against insect pests and pathogens to the host.


Results
In the present study, 22 endophytic fungi were isolated from C. gigantea and screened for inhibitory activity against α-glucosidase and α-amylase. Six cultures exhibited α-glucosidase inhibitory activity in the range of 55-93.4% with maximum being found in AKL-3 (93.4%) followed by AKL-9 (84.4%). AKL-3 also inhibited α-amylase to the extent of 32% while other cultures did not show inhibition against α-amylase. Culture AKL-3 was selected for further studies and identified according to standard taxonomic key including colony diameter, color and morphology of hyphae and conidia. The colonies were slow growing having a diameter of 5.3 cm when incubated on Potato Dextrose Agar (PDA) plates at 30 °C for 9 d. These were white in color when young and turned greenish on maturity with dark reverse (Fig. 1a). Hyphae were septate and branched in the apical region, conidia were multi-celled with transverse as well as longitudinal septa and round to oval in shape. Longitudinal septa were fewer in number than transverse septa (Fig. 1b,c). The genetic relationship of AKL-3 was determined by amplification of ITS1-5.8S-ITS2 rDNA region. The size of the amplified sequence was 476 bp. After sequencing, the sequence was deposited with GenBank under accession number MH071380. Alignment with homologous nucleotide sequences, revealed the strain AKL-3 to be closest to Alternaria destruens with a similarity of 100% with type specimen (Fig. 2). Thus, on the basis of molecular and morphological analysis, the strain AKL-3 could be identified as A. destruens. The culture was submitted to National Centre for Microbial Resource (NCMR) at National Centre for Cell Science (NCCS), Pune, Maharashtra, India under accession number MCC1666.
Column chromatography of ethyl acetate extract of A. destruens AKL-3 yielded two active fractions (AF1 and AF2) which differed with respect to their color and also exhibited different TLC profiles. AF1 was yellow in color whereas AF2 was red. Active fraction AF1 inhibited α-glucosidase enzyme to an extent of 87.75% whereas AF2 showed 72.11% inhibition. Active fractions AF1 and AF2 were also assayed for their inhibitory potential against α-amylase and β-glucosidase. It was observed that AF1 was highly specific as it possessed α-glucosidase inhibitory potential but showed no inhibition against the other two enzymes (α-amylase and β-glucosidase), while active fraction AF2 exhibited inhibition against β-glucosidase (54.62%) as well as α-amylase (34.55%). Both the active fractions were found to possess phenolic compounds after staining with Fast Blue B and FeCl 3 .
Insecticidal activity. Preliminary studies to determine the insecticidal potential were carried out on second instar larvae of S. litura by feeding them on artificial diet supplemented with 1.5 mg/ml of AF1, AF2 and after pooling them together. The mean average larval mortality recorded was 19.99, 23.33 and 33.3 percent due to AF1, www.nature.com/scientificreports www.nature.com/scientificreports/ AF2 and pooled fraction, respectively. The effect of pooled fraction was more evident than the individual fractions therefore detailed studies on various parameters viz. larval mortality, larval period, pupal period and total development period were conducted using different concentrations (0.5-2.5 mg/ml) of pooled fraction.
Larval exposure to diet amended with varying concentrations of pooled fraction of A. destruens resulted in total average mortality of 10 to 70 percent as compared to 3.33 percent in control. The larval mortality increased in a dose dependent manner with significant effect at 2.0 and 2.5 mg/ml (F = 13.55, p ≤ 0.001). The mortality rate increased steadily with the increase in feeding duration (Fig. 3). The LC 50 value was determined to be 1.875 mg/ml using probit analysis. Sluggishness and failure of molting were observed prior to larval deaths (Fig. 4). The negative impact of the inhibitors was also observed on growth and developmental parameters of S. litura. Relative to control, the larval period extended significantly by 3.64 and 4.55 days due to 2.0 and 2.5 mg/ml of pooled fraction of A. destruens respectively (F = 41.92, p ≤ 0.001; Table 1). Similarly, the pupal period was also prolonged with significant effect at these two higher concentrations (F = 3.45, p ≤ 0.001). In comparison to control, the development period of S. litura was delayed by 7.89 and 9.19 days when the larvae consumed 2.0 and 2.5 mg/ml of partial purified inhibitor as evident in Table 1. Toxic effects of pooled fraction were also detected on adult emergence as 23.33 percent adults emerged at the highest concentration as compared to 96.67 percent in control.
Sub lethal effects. Sub lethal effects in the form of morphological deformities like larval pupal intermediates as well as pupae with attached larval exuviae, depressed head and unsclerotised cuticle were also manifested in S. litura (Fig. 5a-c). However, in some of the cases pupae were normal but the adults emerged with underdeveloped and crumpled wings (Fig. 6a,b).
Nutritional physiology. Nutritional analysis revealed a significant influence of pooled fraction of A. destruens AKL-3 culture on food utilization efficiency of S. litura (Table 2). A significant decline of 31.96-53.94 percent in relative growth rate (RGR) over control was recorded when fed on diet amended with different concentrations of the pooled fraction (F = 155.43, p ≤ 0.001). It was observed to be a dose dependent effect. Similarly, a significant decrease was recorded in relative consumption rate (RCR), which dropped by 19.24-72.93 percent over control at different concentrations (F = 442.51, p ≤ 0.001). The highest concentration of the inhibitor was found to be  In vivo effects on digestive enzymes. Addition of inhibitory fraction of A. destruens AKL-3 to larval diet significantly decreased α-glucosidase activity in the insect gut. As compared to control, there was 11.49-47.74 percent decline in the level of α-glucosidase activity after 48 hr (F = 262.34, p ≤ 0.001; Table 3). The larvae feeding on the highest concentration of pooled fraction showed 48.67 percent reduction in enzyme activity after 72 hr over control (F = 368.64, p ≤ 0.001). Similar effects were recorded on β-glucosidase. The decrease in enzyme activity was observed in the range of 18.96-42.06 percent after 48 hr (F = 135.46, p ≤ 0.001; Table 4). Although prolonged exposure to pooled fraction did not further drop the level of enzyme activity, but in comparison to control it remained significantly lower (F = 29.53, p ≤ 0.001). Pooled fraction also significantly suppressed the level of α-amylase of S. litura larvae. Larval exposure to amended diet for 48 hr reduced the α-amylase activity by 18.57-25.37 percent at higher concentrations (F = 36.29, p ≤ 0.001; Table 5) and 14.57-30.42 percent after 72 hr in comparison to control larvae (F = 104.73, p ≤ 0.001).
Antifungal activity. Both the active fractions of A. destruens AKL-3 were examined for their antifungal activity against phytopathogens. It was observed that active fraction AF2 was more potent in terms of its antifungal potential. Even at a lower concentration (250 µg/ml) it evinced higher antifungal activity as compared to AF1 (500 µg/ml). The fraction AF1 exhibited antagonist effect only against all tested Alternaria spp. and Cercospora beticola Sacc., producing inhibitory zones in the range of 10-11 mm whereas AF2 inhibited all the test pathogens  www.nature.com/scientificreports www.nature.com/scientificreports/   www.nature.com/scientificreports www.nature.com/scientificreports/ producing higher zones of inhibition (22-44 mm) ( Fig. 7a-g). The results of antifungal activity of AF2 prompted the examination of the spores and mycelial structures of the affected phytopathogens viz. Alternaria brassicicola (Schwein.) Wiltshire, Alternaria mali Roberts., Alternaria alternata (Fr.) Keissl., C. beticola, Cladosporium herbarum (Pers.) Link, Colletotrichum gloeosporioides (Penz.) Sacc. and Fusarium oxysporum Schltdl.. Light microscopic studies demonstrated severe morphological abnormalities such as leakage of cellular material, thinning of hyphae, formation of vesicles, discoloration of hyphae and alteration in the spore morphology caused by metabolites near the inhibition zone. In A. brassicicola pronounced effects as manifested in mycelial breakage, deformed and shrunken spores and hyphal swellings resulting in bulbous structures were seen under light microscope (Fig. 8a,b). Shrunken and distorted spores were also observed in A. alternata and A. mali (Fig. 8c-f). No visible effects on hyphae were observed. In C. beticola and C. herbarum leakage of cytoplasmic content as well as morphological deformities were observed in spores as noticed in A. brassicicola. The treated spores were lightly stained as compared to untreated ones ( Fig. 8g-j). In F. oxysporum reduction in the size of spores was observed (Fig. 8k,l).

Discussion
In the present study endophytes have been isolated from C. gigantea and screened for their α-glycosidase (α-glucosidase and α-amylase) inhibitory potential. Maximum α-glucosidase inhibitory potential was demonstrated by a culture AKL-3, identified to be Alternaria destruens. Although, a number of reports are available on endophytes with ability to produce α-glycosidase inhibitors [37][38][39][40][41][42] , this is the first study reporting the α-glycosidase inhibitory potential of an endophytic A. destruens. The isolate also exhibited good insecticidal and antifungal activity. A. destruens is a dothideomycetous fungus patented as a bioherbicide for controlling dodder species, a serious parasitic weed in the crops 43 , but no reports are available on its bio-control ability against insect pests and pathogens. Inhibitors of α-glycosidases have been recognized as inherent mechanisms of defense against insect pests [23][24][25] , but only a few reports are available on the insecticidal potential of digestive enzyme inhibitors from endophytic fungi 40,42 . Detrimental effects of α-glucosidase inhibitors produced by an endophytic Exophiala spinifera (Nielsen & Conant) McGinnis on S. litura have been documented by Kaur et al. 42 . In this study, partially purified AGIs from A. destruens were biochemically characterized as phenolic compounds after staining positively with Fast blue B and FeCl 3 . High larval mortality induced in the presence of inhibitory fraction could be attributed to phenolic nature of α-glycosidase inhibitory compounds. Detrimental effects of phenolic compounds on insect pests have been documented by Singh et al. 44 and Singh et al. 45 . The AGI's of A. destruens AKL-3 also delayed the overall development period. Delay in the development period of S. litura when fed on diet supplemented with phenolic compounds has been reported by other workers 45,46 . The molting process was also affected under the influence of A. destruens inhibitors, which caused morphological deformities like larval-pupal intermediates, undeveloped pupae and adults with underdeveloped and crumpled wings. It is possible that the www.nature.com/scientificreports www.nature.com/scientificreports/ AGIs obtained in the present study could be affecting the chitinase enzymes involved in the process of molting. Inhibitors of chitinase are known for their insecticidal activity 47 . Effect of inhibitors from A. destruens AKL-3 was also observed on nutritional analysis as it affected all the nutritional indices. Relative consumption and growth rate of larvae feeding on diet containing different concentrations of inhibitory compounds of A. destruens AKL-3 was also significantly reduced as compared to control. As consumption rate is directly proportional to growth rate, low consumption rate indicates antifeedant effects of the inhibitor. A decreased value of ECI indicated that food ingested was being mainly utilized for energy required for detoxification and less was being metabolized to insect biomass. With the increased requirement for energy, major proportion of digested food is utilized to fulfill energy requirement, hence lowering the ECD value. This diversion of energy from biomass production into detoxification reduces the growth 48 . Also, in vivo evaluation on S. litura's digestive enzymes was carried out to determine whether the deleterious effects being observed under the influence of the inhibitors could be mediated by inhibiting digestive enzymes of insect. The in vivo studies corroborated the in vitro results and activity of all the tested digestive enzymes of S. litura was lowered to a significant level in in vivo studies. The inhibitory effects of phenolics on digestive enzymes have been reported in literature. Singh et al. 45   www.nature.com/scientificreports www.nature.com/scientificreports/ (Caliciales: Physciaceae) and found them to inhibit digestive enzymes of mosquito Aedes aegypti L. (Diptera: Culicidae). Campbell et al. 50 documented the inhibitory effects of an AGI castanospermine on disaccharide enzymes of sap feeding insects.
Glycosidase inhibitors have also been reported to possess antifungal activities 51 . Microscopic observations of phytopathogenic fungi treated with active fraction AF2 revealed severe detrimental effects on mycelium as well as on fungal spores. It is possible that inhibitory metabolites attack the cell wall as indicated by morphological alterations in vegetative cells and spores. As previously suggested they could be inhibiting the enzyme chitinase which is involved in processes during fungal growth and has a role in synthesis and extension of cell wall 30 . Therefore, glycosidase inhibitors can induce antifungal effects by affecting the chitinase enzymes 52 . In in vitro studies, it was determined that AF2 also possessed α-amylase and β-glucosidase inhibitory activities, which may be the contributing factors in increasing its antifungal activity. Kim et al. 53 reported the antifungal activity of a β-glucosidase inhibitor which affected the hydrolytic enzymes of fungi.

Conclusion
This is the first report of α-glycosidase inhibitors from endophytic A. destruens. The study reveals the potential of endophytic fungi as sources of α-glycosidase inhibitors with insecticidal and antifungal potential. The present study also demonstrates that α-glycosidase inhibitory potential can be used as strategy for screening of endophytes with bio-control potential against pests and pathogens.

Materials and Methods
Isolation of endophytic fungi. Endophytic fungi were isolated from different parts viz. leaves and stems of healthy C. gigantea plants. After washing with running tap water, the plant parts were thoroughly rinsed with distilled water. Surface sterilization was carried out with 70% ethanol for 1-2 min followed by treatment with 4% sodium hypochlorite solution for 2-3 min. Again the plant parts were rinsed with sterilized distilled water. To ensure surface sterilization, the water obtained after last wash was plated on PDA. The plant parts (5-6 pieces) in the size range of 2-5 mm were inoculated on water agar plates supplemented with chloramphenicol (20 µg/ ml) (HiMedia, Mumbai, India). Plates were incubated at 30 °C for 3-4 days to few weeks till the hyphae emerged. The emerging fungal hyphae from inoculated plant parts, were picked, purified and preserved on PDA slants for further studies 42 .
Production of secondary metabolites. The production was carried out in Erlenmeyer flasks (250 ml) containing 50 ml of malt extract broth (dextrose 2%, malt extract 2%, protease peptone 0.1%, pH 5.5) 42 . The production medium was inoculated with one plug (8 mm diameter) taken from periphery of freshly grown purified culture. The flasks were incubated for 10 days on a rotary shaker at 250 rpm and 30 °C. After 10 days of incubation, 50 ml ethyl acetate was added to each of the flask and extraction was carried out at 120 rpm and 40 °C for 1.5 hr twice. The extracted organic phase was concentrated on rotary evaporator (BUCHI). The concentrated samples were re-suspended in HPLC grade water and used for further studies.
Assay for α-glucosidase and β-glucosidase inhibition. Enzyme activity was determined in a microtiter 96-well plate. Reaction mixture consisting of 50 μl of phosphate buffer (50 mmol/l; pH 6.8), 10 μl of α-glucosidase enzyme (1U/ml) from Saccharomyces sp. (HiMedia) and 20 μl of inhibitory extract was pre-incubated at 37 °C for 5 min. After 5 min, 20 μl of 2 mmol/l pNPG substrate (HiMedia) (prepared in 50 mmol/l phosphate buffer, pH 6.8) was added followed by incubation at 37 °C for 30 min. Termination of reaction was carried out by addition of 50 μl of sodium carbonate (100 mmol/l) 42 . Acarbose was used as a positive control. The breakdown of pNPG into yellow coloured p-nitrophenol was quantified by reading the absorbance at 405 nm. Every experiment was performed in triplicate, along with appropriate blanks. The % inhibition was calculated using the formula: absorbance of control absorbance of sample 100 absorbance of control For the determination of β-glucosidase inhibitory potential same procedure was applied using p-nitrophenyl -β-d-glucopyranoside as substrate.
Assay for α-amylase inhibition. The assay was conducted as described by Nair et al. 54 with slight modification. The assay mixture containing 200 μl of 20 mmol/l sodium phosphate buffer, 40 μl of α-amylase enzyme from porcine pancreas (2 U/ml) (HiMedia) and 40 μl of fungal extract was incubated for 10 min at 37 °C, followed by addition of 50 μl of starch in all test tubes and further incubated for 20 min. The reaction was terminated with the addition of 500 μl DNS reagent and placed in boiling water bath for 5 min, cooled and diluted with 5 ml of distilled water. Absorbance was measured at 540 nm. The control samples were prepared without any fungal extract. Acarbose was used as positive control.
Identification of the producer culture. Selected culture was identified on the basis of morphological and molecular analysis. Morphological studies were conducted using slide culturing as described by Larone 55 . Thin layer PDA (potato dextrose agar) plates were prepared and small blocks were cut. These blocks were placed aseptically onto glass slide and inoculated with fungal culture. Coverslip was placed over it and incubated at 30 °C. The branching pattern, sporulation and arrangement of the hyphae were examined under microscope (OLYMPUS BX 60). www.nature.com/scientificreports www.nature.com/scientificreports/ Phylogenetic analysis. The culture AKL-3 was identified on molecular basis by amplification of ITS1-5.8S-ITS2 rDNA region using primer pair ITS1 and ITS4 by National Centre for Cell Science (NCCS), Pune (Maharashtra), India. Phylogenetic analysis of AKL-3 culture was conducted by NCCS, Pune.
Partial purification and biochemical analysis. For partial purification extract was loaded onto a silica gel (100-200 mesh size) column (2 × 25 cm). The solvent system used was chloroform: ethyl acetate: formic acid in 5:4:1 ratio. Using this solvent system, fractions of 10 ml each were collected and activity was observed in fraction no. 11 and 14 which were designated as AF1 and AF2, respectively. Chemical nature of active fractions was determined using various TLC based biochemical methods using different visualization reagents viz. Dragendroff 's reagent for alkaloids, FeCl 3 and Fast Blue B for phenols, ninhydrin for amine group, p-anisaldehyde for the detection of steroids and terpenoids.
Insecticidal activity. Insect culture. Insecticidal activity was determined on larvae of S. litura. The culture was collected from fields around Amritsar (Punjab), India. It was reared on Ricinus communis L. (Euphorbiaceae) leaves in glass jars (15 × 10 cm) at 25 ± 2 °C and 65 ± 5% relative humidity in the laboratory. Hygienic conditions were maintained by changing leaves regularly until pupation. The emerging pupae were separated and kept in pupation jars (15 × 10 cm) with 2-3 cm layer of moist sterilized sand covered with filter paper. The adult moths on emergence were transferred to oviposition jars in the ratio of (1:2) males and females|. For nourishment of adults, cotton swab dipped in water and honey solution (4:1) was provided daily as food. To facilitate egg laying the oviposition jars were lined with filter paper. On hatching larvae were maintained on artificial diet as recommended by Koul et al. 56 with slight modifications.
Bioassay studies. Insecticidal potential of both active fractions (AF1 and AF2) of A. destruens AKL-3 was evaluated individually as well as after pooling them together, on S. litura at a concentration of 1.5 mg/ml. Pooled fraction of A. destruens AKL-3 induced significantly higher mortality than individual fractions. Therefore, detailed studies on various parameters viz. larval mortality, total development period were conducted using different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 mg/ml) of pooled fraction. Diet without inhibitor was taken as control. The experiment was designed randomly with six treatments including control and five replications per treatment. Six second instar larvae (6 days old) were taken per replication. Plastic containers (4 × 6 cm) were used to rear larvae individually on treated and control diets. The experiment was conducted under controlled temperature and humidity condition of 25 ± 2 °C and 65 ± 5%, respectively 42 . The diet was changed regularly on alternate days and larvae were checked daily for survival. Observations were made on larval mortality, larval period, pupal period as well as adult emergence. Furthermore, observations were recorded on morphological deformities in larvae, pupae and adults.
Nutritional analysis. The gravimetric method given by Waldbauer 57 was used to determine the nutritional indices. For each concentration of inhibitor, twenty five second instar (6 days old) larvae were starved for 3-4 hr and fed on artificial diet amended with 0.5-2.5 mg/ml of inhibitor from A. destruens and unamended diet served as control. Larvae were maintained in plastic containers (4 × 6 cm) containing a known amount of diet. Optimum temperature and humidity of 25 ± 2 °C, and 65 ± 5%, respectively were maintained. After termination of experiment i.e. after 72 hr larvae, residual diet and faecal matter were separated, dried by incubation at 60 °C for 72 hr and weighed. All nutritional indices were calculated using dry weights and therefore 25 second instar larvae and 25 diet samples were dried to a constant weight to determine fresh/dry weight ratios 58 . Nutritional indices were calculated as per Wheelar and Isman 59 by using following formulae: Effect on glycosidase enzymes of S. litura. To determine the activity of α-glucosidases, β-glucosidases and α-amylase in vivo, late second instar larvae were fed on a diet supplemented with different concentrations ranging from 0.5-2.5 mg/ml of the inhibitor as well as control diet for 48 hr and 72 hr. Ten larvae per replication were used for each time interval and the experiment was replicated thrice. Homogenates (1% w/v) were used as extracts and prepared by homogenizing larval midguts (25 mg) in 2.5 ml distilled water followed by transfer to 1.5 ml centrifuge tubes and centrifuged at 13000 g for 20 min at 4 °C. α-Amylase and α-, β-glucosidase enzyme activities of homogenates were then assayed 42 .