Clean – Soil, Air, Water 2010, 38 (8), 771–774 771

Jatin Srivastava1
Dinesh Shukla2
Short Communication
Vishal Chand3
Ram Naraian4 Mycorrhizal Colonization Affects the Survival of
Harish Chandra5
Anant R. Nautiyal6
Vetiveria zizanioides (L.) Nash Grown in Water
Containing As(III)
1
Department of Environmental
Sciences, Chhatrapati Shahu Ji The presence of arsenic (As) in water is of great public concern. Arsenic exists in three
Maharaj University, Kanpur, Uttar
Pradesh, India common valence states viz., As(0) metalloid arsenic, As(III) (arsenite) and As(V) (arse-
2
Department of Biotechnology, nate). Arsenite [As(III)] is the most toxic form among arsenicals which, predominates in
M.I.M.T., Greater Noida, Uttar anaerobic conditions, generally in flooded soils and in the water with high BOD.
Pradesh, India Experiments were conducted to investigate the effect of As(III) on the mycorrhization
3
Department of Biotechnology, in vetiver (Vetiveria zizanioides (L.) Nash) grass in hydroponics. Studies showed significant
Chhatrapati Shahu Ji Maharaj
University, Kanpur, Uttar Pradesh,
alteration in the mycorrhizal colonization in the roots of vetiver exposed to higher
India concentrations of As(III) starting from 1.0, 2.0, 3.0, 4.0 to 5.0 mg/L prepared in 5%
4
Department of Microbiology, Hoagland nutrient solution without addition of phosphate ions. Considerable
Chhatrapati Shahu Ji Maharaj reduction in the mycorrhizal intensity (M) was observed in all the treatment sets as
University, Kanpur, Uttar Pradesh,
compared to the control suggesting a negative impact of the As(III) on the mycorrhizal
India
5
Department of Microbiology, Gayatri
association. Simultaneously, the study also showed that, As(III) is toxic to the vetiver
Institute of Bio-Medical Sciences, plants having mycorrhizal association however plants with non-mycorrhizal (cleansed)
Dehradun, Uttarakhand, India roots were found to be able to survive for a longer period exposed to As(III).
6
High Altitude Plant Physiology
Research Center, H.N.B. Garhwal Keywords: Arsenite; Hydroponics; Mycorrhizal colonization; Mycorrhizal intensity; Vetiveria
University, Srinagar, Garrhwal, zizanioides
Uttarakhand, India Received: November 13, 2009; revised: April 12, 2010; accepted: April 12, 2010
DOI: 10.1002/clen.200900252

1 Introduction mycorrhizae facilitate phosphorus (P) uptake and the translocation

of less mobile soil nutrients by the plants [11, 12]. Enhanced uptake
Arsenic is a toxic, carcinogenic and ubiquitous element occurring in
of As(V), Zn and Cu by V. zizanioides using chelating agents have been
rocks, soil, water and plants [1]. In some areas of Bangladesh and
recently reported by Chiu et al. [13]. Earlier we have reported the
India, the concentration of As in groundwater has exceeded 2.0 mg/L
accumulation of P in vetiver grown in water [10]. P and As are the
[2]. The presence of arsenic in fresh water and wastewater is of great
chemical analogues [14]. Significant literature is available on As(V)
public concern. The toxicity and bioavailability of arsenic and
uptake and the role of mycorrhizae in other plant specie Pteris vittata
arsenic-containing compounds are varied depending upon valence
[15, 16]. The role of mycorrhizae in the uptake of As(III) is necessary to
state of arsenic [3]. Arsenic exists in three common valence states viz.,
investigate. Liu et al. [16] suggested that the role of mycorrhizal
As(0) metalloid arsenic, As(III) arsenite and As(V) arsenate. Arsenite
associations should be considered during the toxic investigations of
[As(III)] predominates in anaerobic conditions that generally prevail
As on the plants. Because of the occurrence of As(III) in natural
in flooded soils and in water with high BOD [4]. V. zizanioides (L.) Nash
surface waters [4] the role of mycorrhizae in the uptake of As(III)
also called as (Crysopogon zizanioides (L.) Roberty), a native grass of
is necessary to investigate. The current study was performed to
India withstands harsh environmental conditions such as drought,
investigate the effect of higher concentrations of As(III) on the
salinity, metal contaminated sites such as coal, cadmium and gold
mycorrhization since only limited information is available on the
mines [5, 6]. Vetiver, owing to its morphological and physiological
removal of anionic metalloids such as arsenic by any fungal organ-
characteristics, strong deep penetrating aerenchymatous root sys-
isms [17] such as mycorrhizae and its impact on the survival of
tem, unusual ability to absorb and tolerate extreme levels of
V. zizanioides (L.) Nash in aqueous conditions.
nutrients [7] and heavy metals [6] attracted researchers for its
possible use in environmental mitigation especially for wastewater
treatment [8]. In addition vetiver has also been reported to contain
arbuscular mycorrhizal fungi (AMF) in its roots [9] rendering it some
sort of immunity against several pathogens [10]. In general
2 Materials and Methods

2.1 Experimental Setup

Correspondence: Dr. Jatin Kr. Srivastava, Faculty Member, Department of
Environmental Sciences, Institute of Biosciences and Biotechnology,
Chhatrapati Shahu Ji Maharaj University, Kanpur 208024, Uttar
Pradesh, India.
E-mail: jks_345@rediffmail.com
The study was conducted during January–March with repetitive
experimentation in outdoors under a plastic sliding roof. The aver-
age ambient temperature was recorded 22
28C and day light flux
was180 mmol m2 s1 at plant level during the experimentation.

ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clean-journal.com

772 J. Srivastava et al.
Table 1. Growth performance of V. zizanioides (L.) Nash on the exposure to As(III).
Treatment set Concentration Root/shoot length
of As(III) (mg/L) ratio in rootsa)
S zero 0.91L
0.01
A1 1.0 0.50M
0.21
A2 2.0 0.53N
0.08
A3 3.0 0.54O
0.19
A4 4.0 0.56P
0.03
A5 5.0 0.51Q
0.05
Values are mean
SD (n ¼ 6). Different superscript (L, M, N, O, P, Q) denotes the significant (P < 0.01) differences in the column.
a)
Roots infected with mycorrhizae.
b)
Roots were cleansed.
c)
No frond was found alive after 20th day.
2.2 Cleansed and Non-cleansed Vetiver
Naturally growing vetiver grass was collected from the wetlands of
rural area of Lucknow district. The plants were brought to the
laboratory and were cleansed with 1% EDTA to remove elemental
impurities and the soil particles followed by the removal of old
woody roots. The plant material (Vetiver) was kept in plastic trays
(Tarson, Inc.) filled with distilled water to get acclimatized with the
ex situ conditions. After a period of 15 days, new roots appeared in
bunches. To ensure the inoculation of mycorrhizae in the roots, a
layer of normal field soil was laid in the trays and continuous air
supply was set with the help of electronic respirators [10]. Non-
mycorrhizae (cleansed) vetiver fronds were obtained from the culms
grown in closed sterilized bottles containing sterilized distilled
water under same set of conditions. For determining the mycorrhi-
zal association, roots were stained according to the method of Philip
and Hayman [18] whereby fresh roots were treated with 10 % KOH at
908C followed by the staining of the treated roots with trypan blue in
lactophenol followed by the incubation in 1 % HCl.
2.3 Exposure of Arsenite to Vetiver Plant
Uniform fronds of vetiver were taken out from the trays (mycor-
rhizae positive) and bottles (cleansed) and were transferred in the
mesocosm prepared with brown plastic bottles (Tarson, Inc.) as per
Bianchini et al. [19]. A set of six bottles was taken for treatments
including the control. Each bottle contained 25 fronds of vetiver. The
bottles were filled with 500 mL solution of different concentrations
of As(III) viz., 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L prepared with required
aliquots of 100 mg/L stock solution of sodium arsenite (Merck, Inc.) in
5% Hoagland nutrient solution without P. These concentrations are
coded as As1, As2, As3, As4 and As5, respectively. The concentration
of As(III) was determined initially spectrophotometrically by the help
of Spectroquant1 (Merck, Inc.). The phosphorous was not mixed to
avoid P and As competition and to ascertain the effects of As(III) on
the mycorrhization. The growth of vetiver was measured on the basis
of root to shoot ratio. The survival percentage is the simple calcu-
lation of living fronds remaining at the final harvesting. The root to
shoot length ratio was calculated only with the newly emerged roots.
2.4 Mycorrhizal Study
The colonization of mycorrhizae in the roots of vetiver grass was
performed with 1 cm long root fragments [20]. Arbuscules and
vesicles as well as dark septate endophytes of mycorrhizae were
observed in the specimens. Frequency class were distributed on
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Clean – Soil, Air, Water 2010, 38 (8), 771–774
Root/shoot length Plant survival Plant survival
ratio in rootsb) in (%)a,c) in (%)b,c)
0.95L
0.90 100 100
0.73M
0.15 <60 100
0.69N
0.35 50 100
0.79O
0.10 30 100
0.62P
0.20 10 70
0.60Q
0.15 – 40
the basis of the percentage of root segments with fungal coloniza-
tion into A (0 to <1%), B (1–10%), C (11–50%), D (51–90%) and E (>90%).
Mycorrhizal intensity describes the abundance of colonization of the
root system. Mycorrhizal intensity (M) in % was calculated as
M% ¼ (95N>90 þ 70N51–90 þ 30N11–50 þ 5N1–10 þ N<1)/N (N is the number
of root segments) [21]. Mycorrhizal frequency (F%) describes the
percentage of root segments with fungal colonization. Data obtained
were statistically analysed with the help of computer aided software
SPSS 13.0 [22].
3 Results
The results of growth performance of mycorrhizae infected and
cleansed vetiver plants are presented in Tab. 1. The difference in
root to shoot length ratio within 10 days of exposure to As(III) was
found varying from control (S) to (As5) treatment with the values
0.91
0.01 and 0.51
0.05, respectively. Other treatment also
showed a significant change (P < 0.01) in the root to shoot length
ratio. The survival of the plants given in Tab. 1 is the number of
healthy individual frond which was 100% in the control however no
frond was found surviving in As5 of As(III) with a continuous
decrease of survival rates from 60 to 10% in the set As1 to As4.
The root to shoot length ratio in cleansed vetiver plants was
0.95
0.9 in control and was found consistent in all the exposed
sets viz., As1 to As5 with the values 0.73
0.15, 0.69
0.35,
0.79
0.10, 0.62
0.20 and 0.60
0.15, respectively. These values
were quite high as compared to infected plants. Survival rate was
100% in As1, As2, As3 and 70% in As4 and 40% in As5. Cleansed
vetiver plants showed high survival rate whereas infected plants
could not able to survive up to second harvesting. Results showed
that the exposure of 20 days of As(III) ranging between 1 and 5 mg/L
was toxic to mycorrhizae infected plants (Tab. 1). The mycorrhizal
colonization was found more in control sets containing no As(III) and
Figure 1. Mycorrhizal colonization in vetiver grass exposed to the various
levels of As(III) in aquatic conditions.
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Clean – Soil, Air, Water 2010, 38 (8), 771–774
Table 2. Mycorrhizal intensity after 20th day in the roots of vetiver exposed
to As(III).
Treatment M% F% Mycorrhizal status
V C
S 26.0 100 þþþ þ to be altered substantially (Tab. 2; Fig. 1) caused by As(III) in growing
A1 11.0 95 þþ þþ
A2 17.0 100 þþ þ medium.
A3 14.0 100 þ 
A4 14.4 85 þ 
As5 18.0 75 þ 
M, mycorrhizal intensity; F, frequency; V, vesicles; C, coils.
least in the roots treated with 5.0 mg/L As(III). The reduction of
mycorrhizal colonization at first and second harvesting is shown
in Fig. 1.
4 Discussion
All fronds of vetiver grass showed mycorrhizal colonization even
after 20th day of treatment. The mycorrhizal frequency (F%) and
intensity (M%) are presented in Tab. 2. Despite having considerable
frequency ranging between 75 and 100 % in the root segments, the
intensity of the mycorrhizae was found to be altered significantly.
Šraj-Kržič et al. [21] suggested that AM colonization remained abun-
dant in plants under flooded conditions which supported our find-
ings of toxic response of As(III) on mycorrhizal association in
hydroponics. The fronds treated with high As(III) 5.0 mg/L was found
to have higher intensity in comparison of other treatment sets. The
status of vesicles and coils of hyphae was also observed (Tab. 2). The
difference in the frequency of mycorrhizal colonization in mycor-
rhizal roots of vetiver was clearly visualized (Fig. 1) in the frequency
of mycorrhizal colonization. Plants forming mycorrhizal association
including vetiver can grow at lower concentrations of arsenic [23,
24]. Chen et al. [23] suggested that arsenicals affect mycorrhizal
colonization in plants at first harvest. In arsenic contaminated water
mycorrhizal association enhances the uptake of As particularly in
the absence of P whereas the As(III) uptake in the cells is mediated by
P carriers [26]. Present study showed that the mycorrhizal plants of
vetiver initially absorb arsenic at higher rates even at higher con-
centration, because of the initial root to shoot ratio [2]. This trend
lasts soon due to the toxic response of As(III) on the mycorrhizal
colonization and intensity. In the non-infected (cleansed) plants, no
initial impact of As(III) toxicity was found because of the higher root
to shoot ratio leading the plants to adsorb As(III) and avoid the
absorbance in to the tissues which is usually mediated by the mycor-
rhizae. The absence of mycorrhization in the cleansed plants might
have led the vetiver grass to survive up to first and second harvest, i.e.
at the 10th and 20th day of experimentation. Mycorrhizal coloniza-
tion reduces the plant growth which is common in AM plants [27] as
in the case of vetiver grass (Tab. 1). Since most hyper-accumulators
are non-mycorrhizal [28] indicating the biomass related uptake of
the toxic metals and metalloids. Arsenicals are assumed to be taken
up by the plant roots through the same membranes transport system
as of phosphate [29, 30]. In the absence of P, arsenic disrupts the
normal functioning of P metabolism which may be the reason of the
death of the vetiver plants in all the treatment sets. Beever and Burns
[25] suggested that fungi increases the uptake of As faster than P that
might be a cause of reduction in the mycorrhizal intensity as
ß 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Mycorrhiza Affects Arsenite Toxicity in Vetiver Plant 773
mycorrhizae play a major role in plant resistance to As [27]. In
general mycorrhizal colonization by Glomus mosseae and Gigaspora
margarita does not get affected by As toxicity whereas it inhibits the
ericoid mycorrhizal fungus Hymenoscyphus ericae [24]. The findings are
also in agreement with the findings of Liu et al. [16, 24] with a
difference in mycorrhizal intensity and frequency which were found
The authors have declared no conflict of interest.
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