Diversity of fungi associated with mangrove legume Sesbania bispinosa (Jacq.) W. Wight (Fabaceae)

D D Anita, K R Sridhar* and R Bhat*

Microbiology and Biotechnology, Department of Biosciences, Mangalore University, Mangalagangotri 574 199, Mangalore, Karnataka, India
sirikr@yahoo.com
* Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, Penang 11800, Malaysia

Abstract

Sesbania bispinosa is one of the naturally growing seasonal wild legumes in coastal habitats of southwest India having high potential as cover crop, mulch, green manure and forage for livestock.  Fungal assemblage and diversity in surface sterilized and unsterilized tissues (root, stem, leaf, pod and seed) of Sesbania bispinosa growing in the mangrove habitat of River Nethravathi, Southwest coast of India has been investigated.

The surface sterilized tissues yielded 25 endophytic fungi with a highest of 14 species in roots. Aspergillus niger was the most dominant endophytic fungus in all tissues (mean: 32.6%). Among the three endophytic fungi confined to roots, Dactylospora haliotrepha was a typical mangrove fungus. Four endophytic Aspergillus spp. were confined only to seeds. The endophytic fungal diversity was highest in roots than other tissues. The similarity of endophytic fungi ranged between 16% (roots vs. pods) and 72% (stem vs. pods). Among the 40 fungi recorded in unsterilized tissues, a highest of 22 species was associated with leaves. Fusarium oxysporum was the most dominant fungus in all tissues (mean: 65.8%). About 50% of the fungi restricted to any one of the unsterilized tissues. The Simpson diversity was highest in roots, while Shannon in leaves. The fungal similarities were ranged between 12% (pods vs. seeds) and 44% (roots vs. stem, stem vs. pods). A mangrove fungus, Kallichroma tethys has been isolated from unsterilized stem, leaves and pods. Except for seeds (p > 0.05), the frequency of occurrence of fungi differed significantly between surface sterilized and unsterilized tissues (p < 0.001 to p < 0.05).

This study provides a baseline data on fungal association with Sesbania bispinosa for future management of different Sesbania landraces for the benefit of agriculture and animal husbandry.  

Key words: Endophytes, fungal diversity, mangroves, Sesbania bispinosa, wetlands, wild legume

Introduction

The wild leguminous shrub, Sesbania bispinosa (Jacq.) W. Wight [(Synonyms: Aeschynomene aculeata Shreber, Aeschynomene bispinosa Jacq., Coronilla aculeata Willd., Sesbania aculeata (Willd.) Poir., Sesbania cannabina (Retz.) Pers.)] of family Fabaceae (Papilionoideae) is usually cultivated as cover crop to improve the soil fertility and to use as fodder for livestock in a wide geographical range. It is widespread in tropical countries: Africa, China, India, Madagascar, Pakistan and Southeast Asia. It is commonly called canicha, danchi, dunchi fibre, prickly sesban, pricky sisham, sesbania pea (English); sesbane (French); canicha, danchi, dhaincha (Hindi); sanô (Tibetan); mrindazia, msalia-Nyuma (Swahili); sano-khangkhok (Thai). It grows well under water logged or unirrigated conditions, tolerant to high temperatures (36-44°C), high soil alkalinity (pH 10) and establishes during rainy season in a wide variety of soils such as loamy, clayey, black and sandy soils (Prasad 1993, World agroforestry center). It has been considered as a valuable green manure crop due to its fast growth, nitrogen fixation (by root and stem nodulation) and fast decomposition rates (Anonymous 1950, Prasad 1993).

Seeds of Sesbania bispinosa (100 kg/ha) in alkaline soils under rain-fed conditions yield green manure or fodder up to 12 t/ha (Prasad 1993). On mulching Sesbania bispinosa in alkaline soil, the pH decreased (9.28 vs. 8.1), while elevated the total organic nitrogen (0.02% vs. 0.5%) (Prasad 1993). The fiber and seeds of Sesbania bispinosa yield gums such as galactomannans, lignins and cellulose (Salpekar and Khan 1997). Although, generally Sesbania bispinosa has not been considered as an important medicinal crop, it possesses several value added medicinal properties (Misra and Siddiqi 2004). Some of the commercially valuable products of Sesbania bispinosa include: food, fodder, fiber, resin and medicine.

Along mangrove wetlands of the River Nethravathi, Southwest coast of India, Sesbania bispinosa established well and widely used as mulch, green manure and fodder. The present study envisaged documenting the fungal assemblage and diversity of Sesbania bispinosa growing at the Nethravathi estuarine wetlands. The fungal composition, vertical distribution and diversity in surface sterilized and unsterilized five tissue classes of mature plants were assessed to compare the endophytic fungi with epiphytic/saprophytic fungi in view of understanding their role in defense and decomposition of Sesbania bispinosa.   

Materials and methods 

Study site

The study site selected was mangrove region of the River Nethravathi, Southwest coast of India (12°50¢27²N, 74°51¢45²E). The mangrove and backwater area covered the seasonal wild legume, Sesbania bispinosa (Jacq.) W. Wight during monsoon and post-monsoon seasons (July-January) (Figure 1).

The seedlings usually establish during July, flowers in October-November and become senescent/dry in December-February. This legume luxuriously grows in paddy fields and other cultivated lands around the mangrove habitats. The farmers dwelling around the mangroves grow this legume or allow naturally established stands as cover crop for plantations (coconut and areca) or mulch/green manure for some commercial crops (paddy, sugarcane and vegetable) and use as fodder for their livestock.

Sesbania 

During the post-monsoon season (November-December 2006), mature plants about 50 m apart were selected, uprooted, brought to the laboratory and processed within four hours duration. Four tissue classes (roots, stem, leaves, tender pods) from each plant were separated and cut into segments of one cm length and washed in distilled water to remove the extraneous matter. From dry pods, ten seeds per plant were selected to evaluate the fungal component. The tissue segments and seeds were surface sterilized using 95% ethanol (1 min), 6% sodium hypochlorite (5 min) and 95% ethanol (0.5 min) followed by rinse in sterile distilled water. The segments and seeds were plated on antibiotic amended (tetracycline, 250 mg/l) 1.5% potato dextrose agar (PDA) medium. The tissues and seeds without surface sterilization were also plated on the antibiotic amended PDA medium. The plates were incubated at 23±2°C up to four weeks at 12 hr light and dark regime. Periodically they were screened for the growth of mycelia or discrete colonies on the medium or on the segments/seeds. The growing mycelial portions were transferred to fresh antibiotic-free PDA medium. The fungi were identified based on the spore morphology and colony characteristics using standard monographs and taxonomic keys.

Data analysis

The percent colonization frequency and mean percent frequency of all fungi and core-group fungi (frequency of occurrence, ≥10%) of surface sterilized and unsterilized tissues were calculated:

Frequency of occurrence (%) = [(Number of segments colonized) ÷ (Total segments screened)] ×100

Mean % frequency of each fungus = (Total % frequency on all tissues) ÷ (Total tissues screened)

Mean % frequency/fungus = (Total % frequency of fungi) ÷ (Total fungi)

Mean % frequency/core-group fungus = (Total % frequency of core-group fungi) ÷ (Total fungi)

The Simpson and Shannon diversities (Magurran 1988) and evenness (Pielou 1975) were estimated for each tissue. Jaccard’s index of similarity was calculated pair-wise among the tissues based on the presence or absence of each fungal species (Kenkel and Booth 1992). Student’s t-test was employed to assess the difference in frequency of occurrence between sterilized and unsterilized tissues (StatSoft Inc 1995).

Results   

Sterilized tissues

Overall, five surface sterilized tissues yielded 25 endophytic fungi (Table 1).

A maximum of 14 species was recovered from root and seed followed by stem (11 species), leaf and pod (8 species) (Figure 2).

 Aspergillus niger was the most dominant fungus (32.6%) in all tissues followed by Fusarium oxysporum (20.9%) and Aspergillus flavus (10.2%). Six fungi belonged to core-group (frequency of occurrence ≥10%) in at least one of the tissues. Seeds consist of a maximum of five core-group fungi (Figure 3).

Among core-group fungi, non-sporulating sp. 1 was dominant only in root and seed. Three fungi were restricted to root (Codinaea assamica, Dactylospora haliotrepha and Trichoderma hamatum), while four to seed (Aspergillus candidus, Aspergillus oryzae, Aspergillus parasiticus and Aspergillus tamarii). The mean frequency of occurrence per fungus was highest in seed followed by leaf tissues (Figure 3). The contribution of core-group fungi was over 75% in all tissues except for root (<50%). The species accumulation curve between root to seed was not steep in total as well as core-group fungi (Figure 4).

Unsterilized tissues

Unsterilized tissues yielded 40 fungi with a maximum of 22 species in leaf followed by pod (21), root (19) and stem (18) (Table 2, Figure 5).

Fusarium oxysporum was the most dominant fungus (65.8%) in all tissues followed by Aspergillus niger (33,8%) and Penicillium chrysogenum (17.3%). Twelve fungi belonged to core-group and dominant in at least in one of the tissues. Seed consists of a maximum of five core-group fungi (Figure 6).

Up to 50% of the total fungi were confined at least to one of the tissues. The mean frequency of occurrence per fungus was highest in seed followed by stem, pod, leaf and root (Figure 6). The contribution of core-group fungi was over 75% in all tissues except for stem (about 50%). There was a steep increase in the species accumulation curve of total fungi than core-group fungi between root to seed (Figure 7).

Diversity and similarity

In surface sterilized tissues, the Simpson and Shannon diversities and Pielou evenness were highest in root followed by seed (Table 3).

The highest similarity of fungi was seen between stem and pod (72%) followed by stem and leaf (64%) and leaf and pod (60%), while it was least between root and pod (16%) (Table 4).

In unsterilized tissues, Simpson diversity was highest in root and Shannon diversity in leaf, while the evenness was highest in root (Table 3). The similarities were ranged between 12% (pod vs. seed) to 44% (root vs. stem and stem vs. pod) (Table 4). Thirteen fungi were common to sterilized and unsterilized tissues (Table 1, 2). Aspergillus flavus, Aspergillus niger, Fusarium oxysporum and Penicillium chrysogenum constitute the top ranked core-group fungi in both treatments. Except for seed (p > 0.05), the percent frequency of occurrence of fungi differed significantly between surface sterilized and unsterilized root (p < 0.05), stem (p < 0.01), leaf (p < 0.001) and pod (p < 0.001).

Discussion  

Mangroves and estuaries of the west coast of India encompass a variety of natural vegetation such as typical mangroves, mangrove associates and salt-tolerant plant species (Rao and Suresh 2001). Several wild legumes have been established in mangrove and estuarine habitats of the mouth of River Nethravathi. Among them, Sesbania spp. and Canavalia are of special significance to farmers for improving the soil fertility. Sesbania spp. (Sesbania bispinosa and S. rostrata) are seasonal, growing during wet season (June-January), while Canavalia cathartica as perennial creeper. Among Sesbania spp., Sesbania bispinosa is dominant in Nethravathi mangrove and estuarine region and widely used by the farmers as green manure and fodder. 

Studies on mycoflora of mangrove vegetation have been initiated very recently (Hyde and Lee 1995) and out of 54 mangrove tree species and 60 mangrove associates, only 55 have been investigated for fungi (Jones and Alias 1996). Usually the decomposing mangrove litter consists of more of ascomycetes than mitosporic fungi (Kohlmeyer and Volkmann-Kohlmeyer 1991). In our study, fungal component of Sesbania bispinosa consists of more of mitosporic fungi than other groups thus corroborates with earlier investigations on foliar and root endophytes of mangroves, mangrove associates and seagrasses (Suryanarayanan et al 1998, Kumaresan and Suryanarayanan 2001, 2002, Devarajan et al 2002, Ananda and Sridhar 2002). Similarly, endophytic fungi of the coastal sand dune vegetation dominated by mitosporic fungi (Beena et al 2000, Maria and Sridhar 2003).

Our study reveals that different tissues of Sesbania bispinosa were highly colonized by endophytic fungi. Surface sterilized tissues possess lesser fungi than unsterilized tissues (25 vs. 40 species) indicate the selection in endophytic fungi. It has been supported by significant difference in frequency of occurrence between sterilized and unsterilized segments of root (p < 0.05), stem (p < 0.01), leaf (p < 0.001) and pod (p < 0.001). However, Aspergillus flavus, Aspergillus niger, Fusarium oxysporum and Penicillium crysogenum were the top ranking or core-group species in sterilized as well as unsterilized tissues. Similarly, 13 fungi were common in sterilized and unsterilized segments indicates their duel role as endophytes and epiphytes/saprophytes.

Endophytic fungi of Pinus sylvestris and Fagus sylvatica showed tissue specificity in whole-stem and xylem (Petrini and Fisher 1988). In our study, some fungi found in many unsterilized tissues were confined to specific tissue as endophytes (e.g. seed: Aspergillus oryzae; root: Scytalidium lignicola, Trichoderma hamatum) or vice versa (root: Alternaria tenuissima). However, even though Aspergilus parasiticus was confined as seed endophyte, it was also known from unsterilized root tissues. Many endophytic fungi did not establish on the PDA medium without surface sterilization reveals competition of epiphytic/saprophytic fungi, which might have suppressed their growth. For instance, seeds without sterilization yielded only seven fungi, while 14 on sterilization (see Table 1, 2). Fusarium oxysporum, as a highly dominant fungus (75.6%) might have contributed for suppression of other fungi in unsterilized seed.

Contribution of core-group fungi is important as they dominate. In the present study, based on their frequency of occurrence, the core-group fungi contribute between 40 and 80% (see Figure 3, 6) and may play a major role in plant defense or in tissue decomposition. Multiple species dominance was seen in all tissues of Sesbania bispinosa except for unsterilized seed (dominated by Fusarium oxysporum, 76%). Multiple species dominance of endophytic fungi was seen in many mangrove and mangrove associate plant species (Acanthus ilicifolius, Acrostichum aureum, Avicennia officinalis, Lumnitzera recemosa, Rhizophora mucronata and Sonneratia caseolaris) (Kumaresan and Suryanarayanan 2001, Ananda and Sridhar 2002, Maria and Sridhar 2003). Similarly, single species dominance in endophytic fungi was also found in mangrove plants (Avicennia marina, Bruguiera cylindrica, Rhizophora apiculata, Rhizophora mucronata and Suaeda maritima) (Suryanaraynan et al 1998, Suryanarayanan and Kumaresan 2000, Kumaresan and Suryanarayanan 2001).

The similarity of fungi in stem vs. leaf/pod and leaf vs. pod was high (41-72%) unlike other tissue pairs (12-32%) tested with an exception of root vs. stem (44%). This reveals that they have a major role in decomposition of Sesbania bispinosa on its senescence. Most of the fungi recovered in the current study resemble the terrestrial fungi except for two typical mangrove fungi. Dactylospora haliotrepha was endophytic in roots (8.9%), while Kallichroma tethys grew on unsterilized stem, leaf and pod segments (2.2-11.2%). Maria and Sridhar (2003) also recovered a typical mangrove fungus, Cumulospora marina in sterilized roots of Acanthus ilicifolius in Nethravathi mangrove (4%). The wild legumes, Canavalia spp. in coastal sand dunes of the west coast of India also comprise of Halosarpheia sp. (3%) as an endophytic marine fungus (Seena and Sridhar 2004). However, up to 13% of endophytic fungi were marine fungi (Monodictys pelagica, Periconia prolifica, Verruculina enalia and Zalerion maritimum) in roots of coastal sand dune non-leguminous plant species (Ipomoea pes-caprae, Launaea sarmentosa, and Polycarpaea corymbosa) (Beena et al 2000). Although terrestrial fungi dominate as endophytes, depending on the impact of salinity, terrestrial and mangrove fungi may involve in decomposition of plant remains in mangrove habitats (Maria and Sridhar 2003, 2004).

The role of endophytes in grasses has been understood better than non-grass endophytes (Hyde and Soytong 2009). The non-grass endophytes are important due to their ability to decrease insect herbivory, increase drought resistance, increase disease resistance and enhance plant growth (Fröhlich et al 2000; Sieber 2007). Therefore, non-grass hosts gain several beneficial traits by the mutualistic association with endophytic fungi. Endophytic fungi have also been recognized as a major source of novel bioactive compounds and secondary metabolites useful in biological control (Strobel 2003). Schulz et al (1999) predicted the production of herbicidally active metabolites by endophytic fungi is up to 2-3 times higher than phytopathogenic and soil fungi. Carroll (1991) opined that fungal endophytes are capable of defending long-lived plants against short-cycle herbivorous insects.

Our study on fungal associates with Sesbania bispinosa poses many questions. What are the roles of endophytic fungi associated with Sesbania bispinosa? Do they protect these seasonal plants against insect herbivory? In fact, insect herbivores are numerous during the monsoon season of the Southwest coast of India. Do these endophytes enhance the drought resistance and disease resistance? Do they also promote growth and longevity of plant species? These questions are pertinent in the interest of domestication of Sesbania bispinosa, a natural legume landrace used as forage for livestock and to enhance the soil fertility. Further studies need to focus on the fungal endophytic flora of different species of Sesbania distributed in various habitats (e.g. wetlands, dry lands, coastal sand dunes) and their bioactive potential. The fungal decomposition of Sesbania bispinosa in mangrove wetlands will be of special interest to understand the rate of turnover and improvement of soil fertility.  

Conclusions  

• Sesbania bispinosa is a seasonal wild legume growing in wetlands in around the mangroves of the Southwest coast of India and used as cover crop, mulch, green manure and fodder for livestock.

  
  

• The surface sterilized tissues (root, stem, leaf, pod and seed) of Sesbania bispinosa yielded 25 endophytic fungi, while unsterilized 40 species of epiphytic/saprophytic fungi. Except for seeds (p > 0.05), the frequency of occurrence of fungi differed significantly between surface sterilized and unsterilized tissues (p < 0.001 to p < 0.05).

  
  

• Aspergillus niger was the most dominant endophytic fungus in all tissues (mean: 32.6%), while Fusarium oxysporum in unsterilized tissues (mean: 65.8%).

  
  

• Dactylospora haliotrepha, typical mangrove fungus endophytic in roots, while Kallichroma tethys, another mangrove fungus recovered from unsterilized stem, leaf and pod. Mangrove fungal association with Sesbania bispinosa ranged between 2.5-4%.

  
  

• The endophytic fungal assemblage consists of more of terrestrial fungi than marine or mangrove fungi corroborate with the earlier studies on mangrove, mangrove associate and halophytic plant species.

  
  

• Although terrestrial fungi dominate as endophytes in this study, depending on the impact of salinity, both terrestrial and mangrove fungi may involve in decomposition of plant remains in mangrove/estuarine ecosystem.

  
  

• Future studies need to focus on the fungal flora of different species of Sesbania distributed in wetlands, dry lands and coastal sand dunes of Southwest coast of India to exploit their bioactive potential.

Acknowledgements 

The authors are grateful to Mangalore University permission to carry out this study at the Department of Biosciences. DDA acknowledges the University Grants Commission, New Delhi, India and Mangalore University, Karnataka, India for the award of Junior Research Fellowship under scheme Research Fellowship in Sciences for Meritorious Students.

References 

Ananda K and Sridhar K R 2002 Diversity of endophytic fungi in the roots of mangrove species on west coast of India. Canadian Journal of Microbiology  48: 871–878

Anonymous 1950 The Wealth of India, Volume 9, Council of Scientific and Industrial Research, New Delhi, India, pp 293–295

Beena K R, Ananda K and Sridhar K R 2000 Fungal endophytes of three sand dune plant species of west coast of India. Sydowia 52: 1–9

Carroll G C 1991 Fungal associates of woody plants as insect antagonists in leaves and stems. In Microbial mediation of plant-herbivore interactions. (Editors: Barbosa P, Krischik V A and Jones C G), John Wiley and Sons, New York, pp 253–271

Devarajan P T, Suryanarayanan T S and Geetha V 2002 Endophytic fungi associated with the tropical seagrass Halophila ovalis (Hydrocharitaceae). Indian Journal of Marine Sciences 31: 73–74

Fröhlich J, Hyde KD and Petrini O 2000 Endophytic fungi associated with palms. Mycological Research 104: 1202–1212

Hyde K D and Lee S Y 1995 Ecology of mangrove fungi and their role in nutrient cycling: what gaps occur in our mind. Hydrobiologia 295: 107–118

Hyde KD and Soytong K 2009 The fungal endophyte dilemma. Fungal Diversity 33: 163–173

Jones E B G and Alias S A 1996 Biodiversity of mangrove fungi. In Biodiversity of tropical marine fungi (Editor: Hyde K D), Hong Kong University Press, Hong Kong, pp 72–92.

Kenkel N C and Booth T 1992 Multivariate analysis in fungal ecology. In: The Fungal Community: its Organization and Role in the Ecosystem (Editors: Carroll G C and Wicklow D T), Dekker, New York, pp 209–227.

Kohlmeyer J and Volkmann-Kohlmeyer B 1991 Illustrated key to the filamentous higher marine fungi. Botanica Marina 34: 1–61

Kumaresan V and Suryanarayanan T S 2001 Occurrence and distribution of endophytic fungi in a mangrove community. Mycological Research 105: 1388–1391

Kumaresan V and Suryanarayanan T S 2002 Endophyte assemblages in young, mature and senescent leaves of Rhizophora apiculata: evidence for the role of endophytes in mangrove litter degradation. Fungal Diversity 9: 81–91

Magurran A E 1988 Ecological Diversity and its Measurement. Princeton University Press, New Jersey.

Maria G L and Sridhar K R 2003 Endophytic fungal assemblage of two halophytes from west coast mangrove habitats, India. Czech Mycology 55: 241–251

Maria G L and Sridhar K R 2004 Fungal colonization of immersed wood in mangroves of the southwest coast of India. Canadian Journal of Botany 82: 1409–1418 

Misra L N and Siddiqi S A 2004 Dhaincha (Sesbania bispinosa) leaves: A good source of antidiabetic (+)-pinitol. Current Science 87: 1507

Petrini O and Fisher P J 1988 A comparative study of fungal endophytes in xylem and whole stem of Pinus sylvestris and Fagus sylvatica. Transactions of the British Mycological Society 91: 233–238

Pielou F D 1975 Ecological Diversity. Wiley InterScience, New York.

Prasad M N V 1993 Bioresource potential of Sesbania bispinosa (Jacq.) W F Wight. Bioresource Technology  44: 251–254

Rao A and Suresh P V 2001 Coastal Ecosystems of the Karnataka State, India. I – Mangroves. Karnataka Association for the Advancement of Science, Central College, Bangalore, India.

Salpekar J and Khan S A 1997 5,7,3´,4´-Tetramethoxy Quercetin-3-O-b-D-Galactopyranosyl-(1®4)-O-a-D-Xylopyranoside from Syn. Sesbania aculeata. Asian Journal of Chemistry 9: 272–274

Schulz B, Römmert A -K, Dammann U, Aust H -J and Strack D 1999 The endophyte-host interaction: a balanced antagonism? Mycological Research 103: 1275–1283

Seena S and Sridhar K R 2004 Endophytic fungal diversity of 2 sand dune wild legumes from the southwest coast of India. Canadian Journal of Microbiology 50: 1015–1021

Sieber T 2007 Endophytic fungi in forest trees: are they mutualists? Fungal Biology Reviews 21: 75–89

Statsoft Inc 1995 STATISTICA for Windows. Tulsa, OK, USA.

Strobel G A 2003 Endophytes as sources of bioactive products. Microbes and Infection 5: 535–544

Suryanarayanan T S and Kumaresan V 2000 Endophytic fungi of some halophytes from an estuarine mangrove forest. Mycological Research 104: 1465–1467

Suryanarayanan T S, Kumaresan V and Johnson J A 1998 Foliar fungal endophytes from two species of the mangrove Rhizophora. Canadian Journal of Microbiology 44: 1003–1006

World agroforestry center AgroForestryTree Database: A tree species reference and selection guide         http://www.worldagroforestry.org/sea/Products/AFDbases/af/asp/SpeciesInfo.asp?SpID=1516#Uses

Received 29 September 2008; Accepted 27 January 2009; Published 1 May 2009

Go to top