Arch. Microbiol. 97, 69--72 (1974) �9 by Springer-Verlag 1974

Algicidal Effect of 2,4.Dichlorophenoxy Acetic Acid on Blue-Green Alga Cylindrospermum sp.

P. K. Singh

Central Rice Research Institute, Cuttack, Orissa

l~eceived November 14, 1973

Abstract. The effect of the herbicide 2,4-Dichlorophenoxy acetic acid generally used in agriculture was studied on the nitrogen fixing blue-green alga Cylindrosper- mum sp. The alga could tolerate up to 150 ~g per ml in liquid culture and 100 fxg per ml on agar plates without any inhibitory effect on growth and survival. The maximum tolerance was up to 800 ~g per ml and higher concentrations were lethal.

Key words: Aigieidal Effect -- Tolerance to Herbicides -- Blue-Green Alga -- Cylindrospermum -- Growth Effect -- 2,4-Dichlorophenoxyacetic Acid.

Ex tens ive use of pest ic ides and herbicides in agr icul ture has given rise to the p rob lem of thei r effect on soil microorganisms. Blue-green algae are among the ma jo r beneficial n i t rogen fixing microorganisms in the soils used in rice cul ture (Singh, 1961 ; S tewar t , 1970). I t is, therefore, desirable to know the effect of some commonly used herbicides on thei r surv iva l and growth. There are only a few repor ts on the to lerance of b lue-green algae towards these chemicals under defined condit ions. B a t t e r t o n et al. (1971) r epo r t ed the to lerance of Anacyst is nidulans to- wards dieldrin, endrin, a ldr in and the i r metabo l i t e s and t h e y fur ther r epo r t ed t h a t t ox i c i t y of D D T is enhanced b y NaC1 in the m e d i u m (Ba t t e r ton et al., 1972). The to lerance of var ious blue-green algae to- wards pest ic ides genera l ly used in I n d i a has been observed (Venkatara- m a n and R a j y a l a k s h m i , 1972; Singb, 1973).

The p resen t s t u d y was in i t i a t ed to de te rmine the effect of the her- bicide 2 ,4-Dichlorophenoxy acet ic ac id commonly used in agr icul ture on growth and surv iva l of the blue-green alga Cylindrospermum sp. in l abora to ry .

Mater ia ls and Methods

The blue-green alga Cylindrospermum sp. was used in the experiments. The algal filaments were short of average 12 cells with terminal heteroeysts and formed fine suspension in liquid culture. The average length and width of cells measure 3.4 ix and chain of subterminal spores were formed (Singh et al., 1972). I t was grm~a in modified Chu-10 medium (Safferman and Morris, 1964) with trace elements (Allen and Arnon, 1955) under daylight fluorescent tubes for 10 h per day at a light inten- sity of 1000 Lux at 25 =t= 1 ~ C in a culture room. Corning glass vessels and medium

70 P.K. Singh

lacking combined nitrogen (--N medium) were used in all experiments. The cultures were hand shaken twice a day and growth was measured in terms of optical density of culture with a Klett Summerson Photoelectric colorimeter using red filter giving an approximate spectral range of 640--700 nm. The number of filaments, cells and heterocysts were counted by Hemaeytometer counting chamber and growth rate constant was calculated from the equation Kt ~ log10 (Nt/NO) where K ~ growth rate constant, t ~ 8 days, Nt ~ cell number at time t, NO ~ cell number at time 0 (Kratz and Myers, 1955).

Sodium salt of 2,4-Dichlorophenoxy acetic acid (2,4-D) containing 78~ of acid equivalent was used in experiments and its solution was prepared in sterilized medium. Exponentially growing algae was used as inoculum and final volume of medium including herbicide and inoculum was 6 ml.

The colony forming mutant of Cylindrospermum whose growth was similar to parent was used to determine the survival of alga on nutrient agar plates containing various concentrations of 2,4-D and diluted suspension of exponentially growing culture containing 1.5• 103 filaments was inoculated in each plate. These plates were kept in light in culture room and colonies were counted after 8 days.

Results and Discussion

Although kinetic studies were most ly performed with unicellular

forms, it can be applied in few celled filamentous, suspending alga Cyl indrospermum where most of the cells divide almost homogenously. The differentiat ion of paired incipient in tercalary heterocysts which differentiate from paired cells formed from division of single cell in middle of filaments, regularize the fi laments length due to immedia te separat ion of these underdeveloped heterocysts along with fi laments a t tached with them. The development of incipient heterocysts progressed further and this cycle is repeated, thus uniform ~ o w t h of cells of l imited fi laments size is main ta ined . Therefore, this alga is most suitable among hetero- cystous forms for such studies.

The growth of alga was followed in - - N medium after 8 and 16 days a t various concentrat ions (Table 1). I t showed tolerance to 2,4-D up to

800 ~zg per ml in liquid culture. F i laments or cells were absent in higher

Table 1. Effect of different concentrations of 2,4-D on Cylindrospermum sp.

2,4-D (~zg/ml) Mean cell no. per ml Specific growth Heterocysts on the 8th day a rate constant (K) percentage

Control 6.94 • 106 0.148 16.5 100 7.13 • l0 G 0.149 16.0 300 6.3i x 106 0.143 16.0 400 3.21 • 106 0.106 13.6 500 1.95 • 106 0.079 13.6 600 1.13 • 106 0.049 13.7 800 5.72 • 105 0.009 12.5

1000 0 0 0 1200 0 0 0

a Initial cell No./ml = 4.5 • 105.

9G

BC

7(

6 0 !

~c ul

~ 3 0

2 0

~ 4 0 0 5CIO 6()0 7;0 BOO 9~b CONe, (VG/ML)

Fig. 1

Algieidal Effect of 2,4-Dichlorophenoxy Acetic Acid

I00

71

o2 r O.IB

0.16

0.14

oJz C3

o 0.06

0.06

0,04

0.0~

DAYS Fig. 2

Fig. 1. Survival vMue of Cylindrospermum in various concentrations of 2,4-D on agar plates

Fig.2. Growth of CylindrosTermum in 100 tzg/ml (.), 400 ~g/ml (~), 600 (xg/ml (v), 800 (xg/ml 2,4-D (• and control (o) at interval of 24= h

concentrations. Lethali ty in 1000 and 2000 ag per ml was observed within 24 h of inoculation due to loss of turbidity and lysis of cells. There was no effect on growth up to 150 ~g per ml in liquid culture. The alga survived on agar plates up to 600 ~g per ml. Filaments or cells could not be observed microscopically in 900 and 1200 ag per ml (Fig. l). The growth was followed after addition of chemical at interval of 24 h where growth curve of 100 ag per ml was identical to the control, but growth has decreased gradually in 400 and 600 ~g per mI (Fig. 2). There was less growth in 800 (xg per ml (Fig. 2).

The filaments, cells and heteroeysts were counted at various concen- trations and K values gradually decreased with increasing the concen- trations of chemical (Table 1). Filaments were morphologically healthy at lower concentrations and heteroeysts percentage decreased from 16.5 to 12.5 in 800 gg per ml (Table 1).

The cultures isolated from higher concentrations were replated on agar plates containing higher concentrations of 2,4-D to isolate spontaneously occurring resistant mutants. But no resistant mutants could be obtained.

The 2,4-D is commonly used in agriculture to kill weeds and its effect was more on aerobic microorganisms (Klingman, 1961). The recommended dozes of 2,4-D are 1--40 ppm by weight basis in field (Klingman, 196I).

72 P .K . Singh

The to lerance of the a lga used in the presen t s tudy was fa i r ly high. Therefore, the concent ra t ions genera l ly used in agr icul ture m a y no t affect the growth. However , the to lerance m a y differ under field con- di t ion. Hence, i t is suggest ive t h a t to lerance of var ious blue-green algae towards 2,4-D should be s tud ied fur ther bo th in l a b o r a t o r y and in field condi t ion to reach the definite conclusion. The to lerance of var ious species of Rhizobium to herbicides was found to be different (Kaszubiak , 1966; Gillberg, 1971; Gaur and Misra, 1972) bu t higher t han t h a t of p lan t s (Kl ingman, 1961).

Acknowledgements. The author is grateful to the Director, Dr. S. Y. Padma- nabhan, and Dr. S. Patnaik, Head of Crops and Soil Division, for providing ne- cessary facilities and encouragements.

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blue-green algae. In: Taxonomy and biology of blue-green algae, T. V. Desi- kachary, Ed., pp. 264--268. India: University of Madras 1972

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Dr. P. K. Singh Algologist Central Rice Research Institute Cuttack-753006, Orissa India