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1 Chapter II

2 4. Chapter II: The effect of chromium on arbuscular mycorrhizal and dark septate endophyte fungal associations 4.1. Introduction The past decades have witnessed a rapid growth in industries with an accelerated increase in HM pollution in the biosphere (Gisbert et al., 2000). Even though HMs are present in all environmental compartments in trace levels, its excess presence can be a serious cause of concern as it may lead to severe eco-toxicity resulting in loss of biodiversity and can cause hazardous effect on human health (Shanker et al., 2005). The long term persistence of these metals is another serious threat to the environment (Yadav and Chandra, 2011), as they turn out to be conservative pollutants (Kim et al., 2010). The extent of concern over the adverse effect of HMs may differ, as some are toxic and others are essential nutrients at various magnitudes. Large amount of Cr is used as tanning agent in the leather processing and finishing industry, besides in the production of refractory steel, drilling muds, electroplating cleaning agents, catalytic manufacture, wood preservation and in the production of chromic acid (Shanker et al., 2005). As the uptake of Cr into the leather is not complete and forms insoluble complexes with hydroxyl bridge (Khan et al., 2001), tannery industry forms the major source of Cr contamination (Chandra et al., 2004a). But Cr in trace quantity is an essential element for human metabolism as it is required in glucose metabolism and its deficiency may result in diabetes, arteriosclerosis, growth problems and cataract in humans (Khan et al., 2001). Another aspect of Cr contamination is its oxidation state that is, hexavalent Cr is more toxic than trivalent which precipitates at high ph (Khan et al., 2001) and is mutagenic and carcinogenic (Dixit et al., 2002). Besides Cr, other HMs like lead (Pb) and cadmium (Cd) are also released in to the environment in smaller or larger quantities with the tannery effluents and sludge. Soil micro-organisms especially fungi have the ability to protect the plants against heavy metal stress by improving plant nutrition by the absorption of various useful elements (Medina and Azcon, 2010). Arbuscular mycorrhizal fungi, one of the most beneficial soil fungi which provide a direct link between soil and plant, occur in 80-90% 47

3 of the plant species (Harrison, 1997). Even though the symbiotic association of AM fungi with plants has been known from centuries, the recent years have witnessed its improved application in horticulture, agriculture and various decontamination processes (Ruscitti et al., 2011). Generally AM fungi are seen in the most stressed and nutrient depleted environments (Biro and Takacs, 2007; Khade and Adholeya, 2009; Rao and Tak, 2001). The initial colonizers of heavily polluted soils are reported to be metal tolerant plant species which later tend to develop low mycorrhizal colonization which improves the soil organic content. This improved soil microclimate may improve the extent of mycorrhizal colonization which helps in the improved survival rates of the next generation plants (Regvar and Vogel-Mikus, 2008; Leyval and Joner, 2001; Pawlowska et al., 1996). Dark septate endophytic fungi are also beneficial plant associates, which extensively colonize root cortical cells of plants forming melanized or hyaline hyphae which form microsclerotia or moniliform cells (Muthukumar and Prabha, 2012b, Newsham, 1999). Dark septate endophytes are seen more prevalent in some heavy metal contaminated sites than AM fungi (Deram et al., 2008). Even though the DSE fungi have been associated to stressful habitats (Knaff et al., 2012; Frenot et al., 2005; Korhonen et al., 2004), the effect of heavy metals like Cr has not been investigated in detail. In addition, co-existence of AM and DSE fungi are seen in many stressful ecosystems (Wagg et al., 2008). Therefore, it is important to assess the impact of Cr contamination on AM and DSE fungi, so that further studies could be focussed on the chromate detoxification using these fungal endophytes. With this objective in mind, the purpose of this study was to screen the extent of AM and DSE fungal colonization in a heavily contaminated soil with tannery effluents and sludge Materials and methods Study sites Study sites as mentioned in Chapter I Sampling The roots of the seventy four plants species collected from Site-C and Site-NC (mentioned in Chapter I) were thoroughly rinsed with water to remove adhering soil and other adherants. The roots were fixed in FAA (formalin-acetic acid-70% ethanol: 5:5:90; v/v) 48

4 and later transported to laboratory for further processing. Rhizospheric soil (soil shaken from roots and adjacent to roots) was collected and divided into two halves. One half of the soil sample collected from each plant was packed individually in polythene bags and stored at 4 ºC for spore enumeration. The second half of the soil samples of all the plants of a site were bulked and mixed well to form a composite soil sample. This composite was divided into three sub samples, which was used for assessing soil characteristics Determination of soil characters Soil characters were determined as mentioned in Chapter I Preparation of roots for arbuscular mycorrhizal and dark septate endophye fungal assessment Fixed roots were washed free of FAA, cut into 1-cm long fragments and cleared for 45 min. in 2.5% KOH at 100 C, rinsed in water, acidified for 15 mins in 5 N HCl and stained in 0.05% trypan blue overnight (Koske and Gemma, 1989). Squashes of the stained roots were examined for the presence of fungal structures using an Olympus BX51 microscope ( 400). The percentage of root length colonization was quantified according to the magnified intersection method of McGonigle et al. (1990). The procedure adopted for assessing AM fungal colonization was also used for examining the extent of for DSE fungal colonization. We also determined the AM morphological types of the plant species studied. As only squashed roots were examined, we could not distinguish the intermediate subtypes described by Dickson (2004) Enumeration and isolation of arbuscular mycorrhiizal fungal spores Isolation of spores was done from 25g air dried soil as per the procedure described by Muthukumar and Udaiyan (2000). Uncollapsed intact AM fungal spores were transferred to a glass slide containing polyvinyl alcohol lacto glycerol with or without Melzer s reagent (1:1, v/v) using a wet needle (Schenck and Perez, 1990). Spores were identified based on spore morphology and sub-cellular characters. Spore characters were compared with the original description database established by the Schüβler laboratory ( amphylo/amphylo_species.html) and cultural database of INVAM ( 49

5 Plant and arbuscular mycorrhizal fungal nomenclature Plant nomenclature and authorities are used by Henry et al. (1989) and for AM fungi are those of Oehl et al. (2008) and amphylo_species.html Statistical analysis One-way analysis of variance (ANOVA) was used to test the significance of variation within AM and DSE fungal variables. Variations among the plant species occurring in both sites were assessed using t-test. Pearson s correlation was used to assess the relationship between AM and DSE fungal variables (SPSS, Windows Version 9) Spore numbers were log transformed and percentage data on root colonization was arcsine transformed prior to analysis Results Physico-chemical characteristics of soil samples The physico-chemical characteristics of soil samples are mentioned in Chapter I Occurrence of arbuscular mycorrhizal fungi In the present study, 74 plant species were examined for their association with AM fungi. Out of the 41 plants species collected from Site-C (Table 4.1), only 19 (46%) had AM fungal association. Sixty seven percentage (Acrachne racemosa, Alternanthera tenella, Alternanthera triandra, Blumea oxyodonta, Brachiaria ramosa, Calotropis gigantea, Canna indica, Chromolaena odorata, Coccinia grandis, Eleusine indica, Eupatorium odoratum, Lantana camara, Lindernia anagallis, Mikania scandens, Pennisetum purpureum, Peperomia pellucida, Phyllanthus amarus, Phyllanthus reticulates, Ricinus communis, Scoparia dulcis, Synedrella nodiflora, Vernonia cinerea) of plant roots examined from Site-NC (Table 4.2) had AM fungal structures. The AM fungal colonization was characterized by the presence of hyphae, hyphal coils, arbuscules, arbusculate coils and vesicles. Plant species like Carex pellita, Commelina attenuata, Cyperus rotundus, Eragrostis japonica, Lantana camara, Mikania scandens, Peperomia pellucida and Scoparia dulcis collected from Site-C (Table 4.2) had significantly higher AM colonization when compared with their counterparts in Site-NC 50

6 Table 4.1. Arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal association, AM morphology and spore numbers in plant species from heavily chromium (Cr) contaminated site (C) and non contaminated site (NC) sites Families Plant species Sites Fungal association AM type Spore numbers a Aizolaceae Trianthema portulacastrum L. C AM/DSE Paris 2.67±0.88 Amaranthaceae Alternanthera tenella Moq. C DSE 1.67 ± 1.20 NC AM/DSE Paris 5.00 ± 0.58 Alternanthera triandra Steud. C DSE 2.00 ± 1.00 NC AM/DSE Paris 2.67 ± 0.67 Amaranthus spinosus L. C 2.00 ±1.00 NC DSE 1.67 ±0.33 Amaranthus viridis Pollich ex Moq. C DSE 1.33 ± 0.88 NC DSE 5.00 ± 0.58 Araceae Caladium bicolor Vent. C DSE 1.67 ± 0.67 Colocasia esculenta C DSE 1.33±0.33 NC DSE 2.67±0.88 Asclepiadaceae Calotropis gigantea (L.) W.T.Aiton NC AM/DSE Paris 1.00 ± 0.00 Asteraceae Artemisia japonica Thunb. C DSE 4.33 ± 0.33 Blumea oxyodonta DC. NC AM/DSE Paris 6.33 ± 0.67 Chromolaena odorata (L.) R.M.King & H.Rob. NC AM/DSE Arum 3.67 ± 0.33 Eclipta prostrata (L.) L. C. 1.67±0.67 Eupatorium odoratum L. NC AM/DSE Paris 1.00±0.00 Mikania scandens (L.) Willd. C AM 1.33±0.33 NC AM/DSE Arum 2.00±0.58 Synedrella nodiflora Gaertn. NC AM Paris 1.67±0.67

7 Families Plant species Sites Fungal Spore AM type association numbers a Cannaceae Canna indica Redoute C AM Paris 1.00 ± 0.58 NC AM Paris 2.00 ± 0.58 Capparaceae Cleome viscosa L. C ±1.20 NC DSE 5.00 ± 1.00 Commelinaceae Commelina attenuata B. Heyne ex Steud. C AM/DSE Paris 3.33±1.20 NC DSE 2.67±1.67 Compositae Vernonia cinerea (L.) Less C AM/DSE Arum 2.67±0.88 NC AM/DSE Arum 1.33±0.33 Convolvulaceae Ipomea obscura (L.) Less C DSE 1.67±0.67 Ipomea quamoclit L. C DSE 1.33±0.33 Cucurbitaceae Coccinia grandis (L.) Voigt C AM/DSE Arum 1.00±0.00 NC AM/DSE Arum 2.33±0.67 Cyperaceae Carex pellita Muhl. ex Schkuhr C AM/DSE Arum 1.67 ± 0.67 NC DSE 3.33 ± 1.20 Cyperus rotundus Benth. C AM Paris 2.33±0.67 NC DSE 2.33±0.67 Cyperus stoloniferus Retz. C DSE 1.67±0.33 NC DSE 1.00±0.00 Elaeocarpaceae Muntingia calabura L. C. 3.33±1.20 Euphorbiaceae Phyllanthus amaras Schumach. C DSE 1.00±0.00 NC AM/DSE Paris 2.00±1.00 Phyllanthus reticulatus Peir. C AM Paris 2.33±0.67 NC AM/DSE Paris 3.67±0.33 Ricinus communis L. C AM Arum 1.00±0.00 NC AM Arum 1.33±0.88

8 Families Plant species Sites Fungal association AM type Spore numbers a Nephrolepidaceae Nephrolepis sp. C DSE 3.00±0.58 Onagraceae Ludwigia octovalvis (Jacq.) P.H.Raven C DSE 2.33±0.88 Passifloraceae Passiflora foetida Vell. C AM Arum 3.00±1.00 Piperaceae Peperomia pellucida (L.) Kunth C AM/DSE Arum 1.00±0.00 NC AM/DSE Arum 3.67±0.33 Poaceae Acrachne racemosa Ohwi C DSE 1.67 ± 0.67 NC AM/DSE Arum 1.33 ± 0.88 Brachiaria ramosa (L.) Stapf NC AM/DSE Paris 4.67 ± 0.67 Cynodon dactylon (L.) Pers. C AM/DSE Paris 6.33±0.33 Digitaria radicosa (J.Presl) Miq. C DSE 3.33±1.20 Eleusine indica Steud. NC AM/DSE Arum 2.33±0.67 Eragrostis japonica Trin. C AM Paris 1.00±0.00 Pennisetum purpureum Schumach. NC AM/DSE Paris 2.33±0.67 Polygonaceae Persicaria chinensis (L.) Nakai NC DSE 1.67±0.33 Pteridaceae Acrostichum speciosumwilld. C AM/DSE Intermediate 3.00 ±1.15 Scrophulariaceae Lindernia anagallis (Burm.f.) Pennell C DSE 3.67±0.88 NC AM/DSE Paris 1.67±0.33 Lindernia crustacea (L.) F. Muell. C AM/DSE Arum 2.00±1.00 Scoparia dulcis L. C AM Paris 2.67±0.88 NC AM/DSE Paris 1.67±0.67 Solanaceae Datura stramonium L. C AM Arum 1.67±0.67 Physalis minima L. NC DSE 3.33±0.88 Solanum nigrum L. C DSE 2.67±0.88

9 Families Plant species Sites Fungal association AM type Spore numbers a Thelypteridaceae Macrothelypteris torresiana (Gaudich.) Ching C DSE 1.33 ± 0.33 Urticaceae Pouzolzia auriculata Wight NC DSE 2.00 ± 1.00 Verbenaceae Lantana camara L. C AM/DSE Paris 3.00±1.00 NC AM/DSE Paris 2.67±0.67 Woodsiaceae Diplazium esculentum (Retz.) Sw. C DSE 1.67±0.33 a Spore numbers/100g of soil

10 Table 4.2. Extent of arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal in plants species collected from chromium contaminated site (Site-C) Plant name AM colonization (%) a DSE colonization (%) b RLH RLHC RLA RLAC RLV RLT DSEH DSEMI DSEMO DSET Acrachne racemosa ± ± ± 1.15 Acrostichum speciosum 25.55± 2.72 c ± ± ± ± ± ± 1.35 Alternanthera tenella ± ± ± 4.98 Alternanthera triandra ± ± 3.71 Artemisia japonica ± ± ± 1.15 Amaranthus spinosus Amaranthus viridis ± ± ± 3.71 Caladium bicolor ± ± 1.76 Canna indica _ ± 1.33 _ 5.72 ± ± ± Carex pellita ± ± ± ± ± ± ± 1.79 Cleome viscosa Coccinia grandis 11.73± ± ± ± ±0.93 Colocasia esculenta ± ± ± 0.67 Commelina attenuata ± ± ± ± ± ± 3.49 Cynodon dactylon ± ± ± ± ± ± 4.75 Cyperus rotundus ± ± ± Cyperus stoloniferus ± ± 1.76 Datura stramonium ± ± ± ± Digitaria radicosa ± ± ± 6.31 Diplazium esculentum ± ± 1.33 Eclipta prostrata Eragriostis japonica ± ± ± ± Ipomea obscura ± ± ± 0.84 Ipomea quamoclit ± ± 0.99

11 Plant name AM colonization (%) a DSE colonization (%) b RLH RLHC RLA RLAC RLV RLT DSEH DSEMI DSEMO DSET Lantana camara ± ± ± ± ± ± 3.50 Lindernia anagallis ± ± 2.31 Lindernia crustacea ± ± ± ± ± ± 1.43 Ludwigia octovalvis ± ± ± 0.59 Macrothelypteris torresiana ± ± ± 2.20 Mikania scandens ± ± ± ± Muntingia calabura Nephrolepis sp ± ± 1.76 Passiflora foetida ± ± ± ± Peperomia pellucida ± ± ± ± ± 1.11 Phyllanthus amarus ± ± 1.67 Phyllanthus reticulatus ± ± ± Ricinus communis ± ± ± Scoparia dulcis ± ± ± ± Solanum nigrum ± ± ± 1.74 Trianthema portulacastrum ± ± ± ± ± ± 2.32 Vernonia cinerea ± ± ± ± ± 2.71 a RLH - root length with hyphae, RLHC -root length with hyphal coils, RLA - root length with arbuscules, RLAC -root length arbusculate coils, RLV -root length with vesicles, RLT- root length with total colonization b DSEH- root length with dark septate fungal hyphae, DSEMI- root length with moniliform hyphae, DSEMO-root length with microsclerotia, DTC- root length with total colonization c Mean ± S.E

12 irrespective of the high Cr content in the soil. Fungal structures were absent in Carex pellita, Commelina attenuata and Cyperus rotundus collected from Site-NC, whereas, the same plant species were mycorrhizal in Site-C. Arbuscular mycorrhizal fungal structures were absent in the roots of Acrachne racemosa, Alternanthera tenella, Alternanthera triandra, Artemisia japonica, Amaranthus spinosus, Amaranthus viridis, Caladium bicolor, Cleome viscosa, Colocasia esculenta, Cyperus stoloniferus, Digitaria radicosa, Diplazium esculentum, Eclipta prostrata, Ipomea obscura, Ipomea quamoclit, Lindernia anagallis, Ludwigia octovalvis, Macrothelypteris torresiana, Muntingia calabura, Nephrolepis sp., Phyllanthus amarus and Solanum nigrum from Site-C. Roots of A. spinosus, A. viridis, Cleome viscosa, Colocasia esculenta, Cyperus stoloniferus lacked AM fungal structures in both the sites. Out of the four ferns (Acrostichum speciosum, Diplazium esculentum, M. torresiana, Nephrolepis sp.) examined from Site-C, only roots of Acrostichum speciosum contained AM fungal structures. All the members of the Poaceae examined from both the sites, except for Acrachne racemosa and Digitaria radicosa from Site-C were mycorrhizal. Eragrostis japonica showed a colonization rate of 52% even in Site-C. Although 50% of the taxa from the predominant family Amaranthaceae were mycorrhizal at Site-C they were non-mycorrhizal at Site-NC (Table 4.3). Eclipta prostrata and Artemisia japonica (Site-C) lacked AM fungal association among Asteraceae Extent of arbuscular mycorrhizal fungal colonization The different AM fungal variables exhibited significant variations among plant species. The percentage of total root length colonization (RLT) ranged from 8.87 (Cynodon dactylon) to (M. scandens) in Site-C (Table 4.2) and 7.70 (Blumea oxyodonta) to (Ricinus communis) in Site-NC (Table 4.3). The extent of AM fungal structures including percentage root length with hyphae (RLH, F 40,82 =144.69), hyphal coils (RLHC, F 40,82 =115.93), arbuscules (RLA, F 40,82 =246.98), arbusculate coils (RLAC F 40,82 =58.81) and vesicles (RLV, F 40,82 =20.95) significantly (P<0.01) varied among plant species at Site-C. In Site-C, RLH, RLHC, RLA, RLAC and RLV ranged between (L. camara) and (R. communis), 3.79 (Cynodon dactylon) and (S. dulcis), 3.10 (Carex pellita) and (Cyperus rotundus), 3.08 (Acrostichum speciosum) and 51

13 Table 4.3. Extent of arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal colonization in plant species collected from non contaminated site (Site NC) Plant species AM colonization (%) a DSE colonization (%) b RLH RLHC RLA RLAC RLV RLT DSEH DSEMI DSEMO DSET Acrachne racemosa ± 0.87 c ± ± ± ± ± ± 4.44 Alternanthera tenella ± ± ± ± ± ± ± 3.86 Alternanthera triandra ± ± ± ± ± ± 1.48 Amaranthus spinosus ± ± 0.35 Amaranthus viridis ± ± ±1.45 Blumea oxyodonta ± ± ± ± ± 1.76 Brachiaria ramosa ± ± ± ± ± ± ± 2.53 Calotropis gigantea ± ± ± ± ± ± 2.14 Canna indica ± ± ± Carex pellita ± ± 1.76 Chromolaena odorata ± ± ± ± ± ± 1.62 Cleome viscosa ± ± ± 3.27 Coccinia grandis ± ± ± ± ± ± 3.13 Colocasia esculenta ± ± 1.15 Commelina attenuata ± ± ± 2.94 Cyperus rotundus ± ± ± 1.76 Cyperus stoloniferus ± ±1.15 Eleusine indica ± ± ± ± ± ± 1.07 Eupatorium odoratum ± ± ± ± ± ± 1.33 Lantana camara ± ± ± ± ± ± ± 2.93 Lindernia anagallis ± ± ± ± ± ± 0.93 Mikania scandens ± ± ± ± Pennisetum purpureum ± ± ± ± ± ± 1.92

14 Plant species AM colonization (%) a DSE colonization (%) b RLH RLHC RLA RLAC RLV RLT DSEH DSEMI DSEMO DSET Peperomia pellucida ± ± ± ± ± 1.61 Persicaria chinensis ± ± 1.76 Phyllanthus amarus ± ± ± ± ± ± 3.26 Phyllanthus reticulatus ± ± ± ± ± ± 0.57 Physalis minima ± ± 1.15 Pouzolzia auriculata ± ± ± ± 2.06 Ricinus communis ± ± ± Scoparia dulcis ± ± ± ± ± ± 0.16 Synedrella nodiflora ± ± ± ± Vernonia cinerea ± ± ± ± ± 2.00 a RLH - root length with hyphae, RLHC -root length with hyphal coils, RLA - root length with arbuscules, RLAC -root length arbusculate coils, RLV - root length with vesicles, RLT- root length with total colonization b DSEH - root length with dark septate fungal hyphae, DSEMI - root length with moniliform hyphae, DSEMO - root length with microsclerotia, DTC - root length with total colonization c Mean ± S.E

15 26.04 (E. japonica) and 3.17 (Lindernia crustacea) and (Acrostichum speciosum) respectively (Figure 4.1). Likewise, at Site-NC also, the RLH (F 32,66 =85.54), RLHC (F 32,66 =36.39), RLA ((F 32,66 =55.37), RLAC (F 32,66 =121.41) and RLV (F 32,66 =15.90) showed significant variations (P<0.01) among the plant species and ranged between (Coccinia grandis) and (P. pellucida), 5.27 (Pennisetum purpureum) and (Alternanthera triandra), 5.06 (Coccinia grandis) and (R. communis), 1.94 (B. oxyodonta) and (Canna indica), 2.52 (Scoparia dulcis) and 7.66 (Eleusine indica) respectively. Significant variation was seen in all the AM fungal variables for plant species occurring at both the sites except for the RLA (t 2,4 =0.66, P>0.05), RLH (t 2,4 =0.46, P>0.05) and RLT (t 2,4 =0.94, P>0.05) of Coccinia grandis, RLHC (t 2,4 =0.95, P>0.05) of Canna indica and RLV (t 2,4 =0.82, P>0.05) of M. scandens (Table 4.4) Arbuscular mycorrhizal morphological types Almost all the mycorrhizal plants (99%) collected from both the sites had Arum or Paris type AM morphology except Acrostichum speciosum collected from Site-C (Table 4.1). This member of Pteridaceae family had intermediate type morphology. Typical Paris type morphology was more prevalent in Site-C Arbuscular mycorrhizal fungal spores Low density of AM fungal spores was observed in the rhizospheric soil of all the 74 plant species collected from both the sites (Site-C and Site-NC). Both the sites harboured only a single spore morphotype belonging to Scutellospora calospora. There was not much difference in the number of spores of each plant species from both the sites as AM fungal spores ranged between 1 (Canna indica, Coccinia grandis, E. japonica, Peperomia pellucida, Phyllanthus amarus, R. communis) to 6 (Cynodon dactylon) per 100 g of soil at Site-C and 1 (Calotropis gigantea, Cyperus stoloniferus, Eupatorium odoratum) to 6 (B. oxyodonta) per 100 g of soil at Site-NC (Table 4.1). Significant (P<0.01) variation was seen in spore numbers among plant species at both Site-C (F 40, 82 =2.08) and Site-NC (F 32, 66 =3.51). 52

16 Table 4.4. Results of t-test for plant species occurring in both chromium contaminated and non contaminated sites Plant species d. f. Arbuscular mycorrhizal fungi a Dark septate fungi b RLH RLA RLHC RLAC RLV RLT DSEH DSEMO DSEMI DSET Acrachne racemosa t 2, *** -5.46** ** *** 3.19* 4.00** -6.01** 0.59 Alternanthera tenella t 2, ** -6.61** -6.08** -7.30*** ** -2.53* 2.65* Alternanthera triandra t 2, *** *** *** ** 0.53 Amaranthus spinosus t 2, *** *** Amaranthus viridis t 2, * Canna indica t 2, *** 5.06* -9.23*** Carex pellita t 2,4 8.55*** 4.81** *** 9.96*** 6.78** *** 8.32*** Cleome viscosa t 2, *** ** -16.4*** Coccinia grandis t 2, Colocasia esculenta t 2, ** 5.00** * Commelina attenuata t 2, *** 2.83* 3.43* 24.08*** 7.19*** 5.56** -2.44* Cyperus rotundus t 2, *** 28.95*** *** -18*** *** *** Cyperus stoloniferus t 2, ** ** Lantana camara t 2,4-3.56** -4.55** ** -4.72** 8.82*** 9.54*** -2.60* 10.55*** Lindernia anagallis t 2, *** *** -4.47** *** 6.29** ** Mikania scandens t 2,4 5.16** 22.38*** *** Peperomia pellucida t 2,4-2.73* 2.07* Phyllanthus amarus t 2,4-4.03* 6.94** *** * * Phyllanthus reticulatus t 2, ** -5.12** -4.05** -7.63*** 2.42* * Ricinus communis t 2,4 3.76** -6.31** * Scoparia dulcis t 2, * 2.65* 3.67* 9.24*** *** *** Vernonia cinerea t 2,4-2.62* -2.52* * a RLH, RLA, RLHC, RLAC, RLV and RLT indicates percentage root length with arbuscular mycorrhizal hyphae, arbuscules, hyphal coils, arbusculate coils, vesicles and total colonization respectively b DSEH, DSEMO, DSEMI and DSET indicates percentage root length with dark septate endophte fungal hyphae, microsclerotia, moniliform cells and total colonization respectively *, **, *** Significant at P<0.05, P<0.01 and P<0.001 respectively

17 Figure 4.1. Arbuscular mycorrhizal colonization in plant species growing on chromium contaminated soil: a. Appresorium (arrow head) in Vernonia cinerea b. Arbusculate coils (ac) in Commelina attenuata c. Arbuscules (ar) in Lantana camara d. vesicles (v) in Alternanthera tenella e. hyphal coils (hc) and vesicles (v) in Eragriostis japonica. Scale bars: a=100 μm; b-e=50 μm.

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19 Occurrence and extent of dark septate endophyte fungi Of the 74 plant species examined for DSE association 88% from Site-NC and 68 % from Site-C had DSE fungal association (Table 4.1). Only 28 of the 41 plant species examined from Site-C had DSE fungal structures (Table 4.1). Except Canna indica (Cannaceae), M. scandens (Asteraceae), R. communis (Euphorbiaceae) and Synedrella nodiflora (Solanaceae), all the other plant species collected from Site-NC had DSE fungal association. Canna indica, M. scandens and R. communis occuring in both the sites lacked DSE fungal association. Moderate rate of DSE colonization was seen in Amaranthus viridis, Alternanthera tenella, Carex pellita, Cyperus stoloniferus, Lantana camara, Lindernia anagallis, and Phyllanthus amarus collected from Site-C while the same plant species had low or absence of DSE fungal structures at Site-NC. Dark septate endophytic fungal microsclerotia were found only in 16 plant species, of which 5 plants (Amaranthus viridis, Carex pellita, Commelina attenuata, M. torrensiana, S. nigrum) belonged to Site-C. The roots of A. racemosa, Alternanthera tenella, Alternanthera triandra, Cleome viscosa, Cyperus rotundus, Commelina attenuata, and L. camara collected from Site-NC contained microsclerotia, whereas microsclerotia were absent in many of the DSE fungal associated plants in heavily polluted Site-C. Moniliform cells were seen in roots of 22% of plants examined from Site-C and 15% plants examined from Site-NC. The percentage of total root length colonized by DSE fungi (DSET) ranged from 6.56 (Peperomia pellucida) to 72 (D. radicosa) and 5.74 (S. dulcis) to (Cleome viscosa) procured from Site-C and Site-NC respectively (Table 4.2 and 4.3). Similarly, the percentage root length with DSE fungal hyphae (DSEH) ranged from 5.74 (Peperomia pellucida) to (D. radicosa) in Site-C and 5.74 (S. dulcis) to (Cleome viscosa) in Site-NC (Figure 4.2). In both the sites, the minimum percentage root length with microsclerotia (DSEMI) and moniliform cells (DSEMO) were seen in Cleome viscosa (2.61) and Pouzolzia auriculata (1.75) collected from Site-NC (Figure 4.2). Likewise, the maximum %DSEMI (11.89) and %DSEMO (25.72) were observed in Commelina attenuata and Cynodon dactylon examined from Site-C. Significant (P<0.01) variation existed for DSEH, DSEMI, DSEMO and DSET among plant species in Site-C 53

20 Figure 4.2. Dark septate endophyte fungal colonization in plant species growing on chromium contaminated soil: a. Dark septate endophytic fungal hyphae (dh) in Artemisia japonica b. DSE fungal hyphae (dh) and microsclerotia (ms) in Chromolaena odorata c. microsclerotia (ms) in Commelina attenuata d. microsclerotia (ms) in Cleome viscosa. Scale bars: a-d= 50 μm.

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22 (F 40,82 =86.62, F 40,82 =28.26, F 40,82 =37.00, F 40,82 =93.8) and Site-NC (F 32,66 =48.19, F 32,66 =10.61, F 32,66 =10.04, F 32,66 =47.07) respectively. Significant negative correlation existed between RLT and DSET at Site-C (r= ; P<0.01; n=41). Spore numbers and DSEMO also showed significant positive correlation at Site-C (r=0.517; P<0.01; n=41). 4.4 Discussion The tannery effluent contaminated soil had high concentrations of Cr, which is a clear indication of the extreme selection pressure on the AM and DSE fungal endophytes. The incomplete uptake of Cr during tanning explains for the presence of high Cr content in the highly contaminated soil. On a physico-chemical examination of tannery effluent laden soil, Verma and Biswas (2013) reported the alkaline nature of contaminated soil ( ) which falls in line with the present study. The high levels of N, P and K in Site-C could be attributed to the organic content present in the tannery sludge (Munn et al., 2000). There are also reports on the presence of N, Ca, Mg, P and small amounts of sodium in tannery effluents (Ogbonna et al., 1998; Frank, 1998). Less than half the percentage of total plants examined from Site-C had AM fungal association in spite of the fact that high concentration of HMs are known to adversely affect the AM fungal formation and function (Del Vel et al., 1999; Pichardo et al., 2012). The adverse effects of arsenic on AM fungal colonization were reported in Nicotiana tabacum by Jianfeng et al. (2009). Likewise, low levels of AM colonization was seen in multi metal (Cu, Zn and Mn) contaminated soil inoculated with indigenous (31%) or introduced (19%) AM fungi (Wang et al., 2005). Maximum AM root colonization of 35% was reported for clover growing on mine spoils heavily contaminated with Zn and Cd (Gildon and Tinker, 1981). In a calamine spoil mine rich in Pb and Zn in southern Poland, only 17% of the absolute cover was harboured with mycorrhizal plant species (Pawlowska et al., 1996). In the present study, the high nutrient levels in contaminated soil could also be a reason of lack of AM colonization in plant species, as soils with high fertility tend to decrease the dependence of plants on AM fungi (Siqueira and Saggin - Junior, 1995). Nevertheless, irrespective of the high Cr concentration in the soil, AM association was present in many plant species suggesting the avoidance or tolerance of AM fungi to 54

23 heavy metal stress (Leyval et al., 1997). High rate of AM colonization was also seen in wheatgrass examined from a subalpine coalmine soil and in oil sand tailings (Zak and Parkinson, 1982). The maximum RTC in Site-C occurred in M. scandens (65.35%) which exceeded the maximum colonization levels recorded in R. communis (61.30%) at Site-NC. This is in line with the findings of Khade and Adholeya (2009) where 100% of the root length was colonized by AM fungi in Parthenium sp growing on a tannery effluent contaminated soil. The highly mycorrhizal plants would have managed to escape the high metal enriched soil micropockets in Site-C as high rate of AM colonization was seen in Agrostis capillaris on a Zn and Cd contaminated site close to a zinc refinery (Griffieon et al., 1994). Oxalis acetosella examined from a multiple HM in (Cd-Zn, Pb) contaminated soil had higher colonization levels than those examined from uncontaminated soil (Turnau et al., 1996). Weissenhorn et al. (1995a, b) also reported high rate of AM colonization in plants collected from soil that received multiple metal contaminations. The slightly alkaline nature of the contaminated soil may also immobilize Cr to certain extent, making it less bioavailable to the plants (Aydinalp and Marinova, 2003) thus reducing its toxicity. The lack of AM colonization in the roots of certain plant species like A. spinosus, A. viridis, Cleome viscosa, Colocasia esculenta, Cyperus stoloniferus in both the sites suggests the influence of host specificity, soil ph, presence, type, availability of AM inoculum and nutrient levels (Friese and Allen 1991; Sanders and Fitter 1992a) other than the role of HM pollution on the formation of AM fungal association. In the present study, the members of Poaceae either had moderate levels of AM fungal colonization or lacked it except for Eragrostis japonica from Site-C. An examination on the mycorrhizal status of plants growing on tannery effluent contaminated soils by Khade and Adholeya (2009), revealed that Cynodon dactylon had higher AM colonization in contaminated than in non contaminated soil (29.50% vs 10%). However, in the present investigation C. dactylon (8.87%) had low rate of AM fungal colonization at Site C. This is contrary to the results of Soares and Siqueira (2008) where grasses were found to be tolerant to HM stress as evidenced by AM colonization rates as shown for Brachiaria 55

24 decumbens in multi-contaminated soil (Zn, Cd, and Cu). Some indications also exist on the significance of AM fungi in the phytoextraction of HMs like Pb from contaminated soil by vetiver grass (Chrysopogon zizanioids) (Pumamiya et al., 2010) Only E. prostrata and Artemisia japonica lacked AM association among the six members examined from Asteraceae. Many hyper accumulating plants are known to occur in Asteraceae and their HM tolerance could be attributed to the rhizospheric microorganisms like AM which helps the plants to absorb nutrient or immobilize the HMs (Turnau and Mesjasz-Przybylowicz, 2003). The predominance of Amaranthaceae members at Site-C could not be attributed to the AM fungi, but to other factors like high organic content and nutrients. The high crop yield due to the improved physico-chemical conditions of soil by the application of tannery effluents was reported by Munn et al. (2000). Even though many studies have reported the role of AM fungi in the hyperacccumulating capacity of pteridophytes like Pteris vittata (Cai et al., 2004; Leung et al., 2006), the absence of AM association in pteridophytes in the present study except Acrostichum speciosum could be because of the high Cr content in the soil. Studies have even shown the enhanced uptake of arsenic in mycorrhizal P. vittata (88.1g Kg -1 ) in comparison with non mycorrhizal types (60.4 mg Kg -1 ) (Leung et al., 2006). In the present study Paris-type was more prevalent at Site-C. Likewise, in a multi metal contaminated site (Zn, Cd, Pb) Paris-type AM morphology was found to be more widespread while in non contaminated site Arum-type was more common (Voges-Mikus et al., 2005). In contrast, a study by Khade and Adholeya (2009) showed the prevalence of Arum-type AM morphology in both tannery effluent contaminated and non contaminated sites. The slow growth rate specific to Paris type AM morphology (Yamato, 2004) is found to be the best strategy for the long term survival of plants growing in high stress environments (Brundrett and Kendrick, 1990). In the present investigation, both the sites harboured only spores of Scutellospora calospora. Reports on single species of AM fungi were also reported from soils which were highly contaminated with Zn, Cu, Pb, Ni, Cd and Mn (Griffioen, 1994). The low species richness in both contaminated and non contaminated soils was also reported by Pawlowska et al. (1996) and Regvar et al. (2001). The spore count decreased 56

25 as the total heavy metal (Zn, Cd, Pb, and Cu) concentration increases in Vertisol and Leptosol soils (Ortega-Larrocea et al., 2001). Low spore numbers were observed in contaminated soils when compared to less contaminated sites (Leyval et al., 1995). In contrary, studies done by Khade and Adholeya (2009) showed higher AM spore diversity in metal contaminated soil in comparison with non contaminated soil. Moreover, the high P content of both the soils (Site-C and Site-NC) could also be attributed for the low species richness (Peng et al., 2010). Elevated levels of Cr resulted in low number of AM spores in sites filled with tannery sludge when compared to Site-NC in the present study. Similarly del Val et al. (1999) also showed that toxicity of heavy metals can reduce the number of spores in the rhizospheric soil. Despite the excessive heavy metal contamination there are several reports on high species diversity of AM fungi in metal contaminated soil like arsenic (Gonzalez-Chavez et al., 2002) and magnesite mine spoils in India (Raman et al., 1993). Low spore density did not affect the percentage colonization of AM fungi in the present study. This corroborates with the findings of Khade and Adholeya (2009), Del Val et al. (1999) and Jacquot-Plumey et al. (2001) where low spore number did not have any negative effect on the development of AM fungal structures. Regvar et al. (2001) and Whitfield et al. (2003) also reported that the AM fungal development never suffered from the less abundant nature of AM fungal spores. This could be due to the favourable environmental conditions for the germination of the AM spores resulting in rapid colonization and low spore number (Khade and Adholeya, 2009) as spore germination is less susceptible than any other facet of AM fungal life cycle (Pawlowska and Charvat, 2007). The significant positive correlation between the spore numbers and DSMO suggests that, the influence of environmental factors on these fungal variables are directly proportional (Muthukumar and Vediyappan, 2010). The high rate of DSE colonization revealed in the plant roots of Site-C demonstrates that these fungal endophytes are tolerable to high Cr content in the soil. This corroborates with the findings of Barrow et al. (1997) where high rate of DSE colonization was seen in dominant shrubs and grasses in arid grasslands of south western USA. Ban et al (2012) and Zhang et al. (2008) isolated DSE fungi from the roots of plant 57

26 species grown in lead-zinc mine tailings and waste smelter site respectively which is a clear indication of its tolerance towards HM stress. Muthukumar and Vediyappan (2010) also reported the presence of DSE fungal colonization in >90% of the plant species examined from pulp and paper mill effluent irrigated soil. Symbiotic association of plants with the dark septate fungal endophytes provides the plants with the tolerance to dwell with various stresses in the soil in comparison to AM fungi (Christie and Kilpatrick, 1992; Muthukumar and Vediyappan, 2010). The significant negative correlation between AM and DSE colonization in the present study falls in line with the findings of Muthukumar and Vediyappan (2010) where an inverse relationship between total root colonization of AM and DSE was reported in the plant species collected from pulp and paper mill effluent irrigated soil. Scervino et al. (2009) also recently reported the reduction of AM fungal development by the exudates of DSE fungi. The exudates of Dreschlera sp. negatively influenced the development of extra matrical phase of the AM fungus Gigaspora rosea (Scervino et al., 2009) Conclusions Understanding the impact of HMs on AM and DSE would be helpful in developing more innovative, cost effective and successful strategies in minimizing metal toxicity on plants and in soil, which could be implemented in various re-vegetation practices. Further studies are necessary in order to confirm the ability of AM and DSE to survive in high HM concentrations, as they could be possible candidates for mycoremediation. 58