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INTERACTIVE MEETING ON THE ROLE OF
AZOLLA
IN CONTROLLING
RICE FIELD BREEDING
VECTORS
Report of an Interactive Meeting
Pondicherry, India
9 April 1999
Vector Control Research Centre
Indian Council of Medical Research
Pondicherry
Introduction
Globally rice is grown in 140 million hectares of land serving as a staple
food. Since rice fields are extensively irrigated they favour the prolific breeding of
many disease vectors. In India about 14 species of mosquitoes belonging to 5 genera
viz. 6 u/ex, Aedes. Anopheles, Armegeres and Mansonia have been suspected or
incriminated from disease outbreak studies as vectors of Japanese encephalitis (JE).
The principal vectors of JE, Cx. vishnui sub-group breeds mainly in rice fields Over
40 viruses have been identified, in rice field agro ecosystems but most important
one is Japanese Encephalitis virus. Though IE was first reported in India only in
fifties, in recent times its incidence has been recorded in as many as 24 states and
union territories. The major and repeated outbreaks of the disease have been reported
from Assam, Andhra Pradesh, Bihar, Goa, Karnataka, Kerala, Madhya Pradesh,
Manipur. Pondicherry. Tamil Nadu, Uttarpradesh and West Bengal Most of these
states are principal rice growing states of the country.
The waterfern, Azolla is being promoted in rice cultivation to reduce the input
cost of nitrogenous fertilizer and also to increase the yield. Azolla is also being used
as feed for live stock, green manure, weed control, ornamental purposes and compost
preparations. It is being used in 2-5% and 40-60% areas of rice cultivation in China
and Vietnam, respectively besides India, Philippines, Japan, Korea, Thailand and
USA. Besides being a green manure, Azolla is also known to acts as a mechanical
barrier to the ovipositing female mosquitoes, since it forms a dense mat on the water
surface. However, its potential as bio control agent has not been fully explored and
hence an interactive meeting was organized to understand the potential of Azolla as
a control agent of mosquito breeding. The resume of the deliberations of the
interactive meeting and conclusions and recommendations are presented hereunder
Mosquito breeding in Rice fields and incidence of Japanese
Encephalitis
For rice cultivation fallow lands are flooded with water, ploughed, levelled
and transplanted with rice seedlings and immediately after transplantation, mosqui
toes start breeding. The density of early instars of both Anophelines and ( ulicines
show maximum values upto 3rd week, and thereafter there is a steady decrease in
the density From the specimens collected from the field, a total of ten Anopheline
mosquitoes viz. An. suhpictus, An. vagus. An. harhirostris, An. hyrcanus, An.
splendidus, An. pallidus, An. fluvialitis, An. jamesi and An. lessellalus have been
recorded in the order of abundance. Five species of ('ulicines viz., ( x. vishnui, ( x
tritaeniorhynchus, Cx. pseudovishnui, Cx. gelidus and Cx. hitaeniorhynchus have
also been recorded in the above study. The peak breeding of JE vectors occurs during
(he first two weeks after transplantation in short-term rice varieties while in long.
L rice varieties it occurs during 6 to S weeks in add,t,on to two peaks dunng first
4 weeks.
Ln South Arcot district of Tamil Nadu, vector abundance started raising from
July and minimum infection rates (MIR) peaked in September followed by a decrease
in MIR during the month of October. However, the abundance of mosquitoes
remained high until March. The decrease in MIR from October onwards coincided
with rising herd immunity in pigs. Although MIRs in October (0.47) and November
(0 42) were lower than in September (0.92), a comparable high risk of infection for
humans continued because of high vector abundance and human biting rates (Ga-
janana et al. 1999)
Azolla and its occurrence
AzoIla is a cryptogenic free floating water fern (SalvinialesMzo//acea).
Although this fem is represented by 31 species, only 6 are existent, viz. A. caroliniana, A. filiculoides, A. mexicana, A. microphylla, A. nilotica and A. pinnata (Lump
kin and Plucknett, 1978). In India the dominating species is represented by A.
pinnata, occurring in ponds, pools, reservoirs, rice fields and lakes (Majid, 1986;
Ansari and Sharma, 1991). The plant body is pinnately branched with free floating
horizontal stem bearing long adventitious roots on its underside leaves that are
arranged alternately overlapping each other; each leaf consists of one dorsal thick
green aerial lobe and a ventral thin colourless lobe, which remains submerged in
water; absorption of nutrients is almost entirely through ventral lobe and the roots
are practically non-functional 1 (Hill, 1975). Anabaena azollae, a cyano-bacterium
lodged in the dorsal lobe cavity and is regarded as source of organic fertilizer for
rice cultivation (Lumpkin and Plucknett. 1980; 1982). The azollae not only fulfils
the nitrogen requirement of host plant Azolla but also liberates substantial amount
of nitrogen in the medium. The carbon requirement of the soil is met from Azolla.
The trophic independence of Anabaena azollae for nitrogen and that of Azolla for
caibon, makes the system complete in all respects, for converting molecular nitrogen
into ammonia.
A SUrvey carned out t0 understand the natural infestation of A. pinnata in
different water bodies (Ansari and Sharma. 1991) revealed that highest infestation
occurred in pools and lowest in paddy fields. Of the 178 pools checked, 36.5% were
complerely covered with the fem growth while 34.8% were partially covered and no
infestation was observed m 28 6% Similarlv 6 w
n '
ut»iv
anH o 9°/rar,
j
•,
.
„ ‘3imilai *5 6.5 /o of the wells were completely
and 9 _/o partly covered withzlzo//a, while 84.2% had no Azolla.
2
Technology for mass Multiplication and Application of
Azolla
Azolla normally reproduces vegetatively by fragmentation. Under optimum
conditions doubling time of Azolla falls between 2 and 5.6 days (Watanabe et al.
1977, Talley etal., 1977; Kannaiyan, 1988b; 1990). The growth potential of Azolla
is subjected to environmental variables such as humidity, temperature, light inten
sity, pH, and salinity (Kannaiyan and Somporn, 1987; Kannaiyan et al., 1987 and
Kannaiyan, 1988a). Since factors controlling the formation and germination of
sporocarps in different species of Azolla are still poorly understood, production of
large scale inoculum has to be done through vegetative multiplication. Further, the
storage and transport of fresh, bulky material is not only cumbersome but also
impracticable as Azolla undergoes rapid decomposition during storage under tropical
conditions.
A simple nursery method for large scale multiplication of Azolla has been
evolved for easy adoption by the farmers (Kannaiyan, 1982) The field selected for
Azolla nursery is thoroughly prepared, and divided into one cent plots (20 X 2m) by
providing suitable bunds and irrigation channels. Water is maintained to a depth of
10cm Ten kg of fresh cattle dung mixed in 20 litres of water is sprinkled in each
plot and Azolla inoculum at 8 kg is applied and spread uniformly. Super phosphate
(100 g) is applied in three split doses at 4 days interval as fertilizer for Azolla. For
insect pest control furadan granules at 100 g is applied 7 days after inoculation.
Fifteen days after inoculation, Azolla is harvested and introduced into the main field
as a source of primary inoculum. From one harvest 40-50 kg of Azolla is obtained
from each plot. Azolla nursery may be planned while rice nursery is raised.
Azolla may be inoculated either before planting rice or 7-10 days after
planting. The inoculum required for one hectare transplanted rice is 500 kg. The
inoculated Azolla is found to establish and cover the rice field in 20-30 days period
which is equivalent to 15-25 tonnes of biomass/ha. (Kannaiyan, 1986) Thisbiomass
is incorporated in the rice field during first weeding which decomposes in 2-3 weeks
period (Subramani and Kannaiyan, 1987) and the nitrogen and other nutrients are
made available to the rice crop. The left overfronds ofAzolla float in water surface.
multiply and cover the rice field again. Likewise, two or three incorporations of
-1
Azolla mat is possible for one rice crop. Azolla contributes 40-60 kg N ha
(Kannaiyan, 1992) besides Phosphorus, zinc and iron.
3
Control of Mosquito Breeding By AzoIla
_
_
Around 1900, a strong international interest developed in the use of Azolla
for mosquito control (Smith, 1910; Lumpkin and Plucknett, 1980). Azolla mats were
thought to prevent mosquitoes from laying eggs and prevent larvae from coming up
for air. This claim was supported by Tan chuan-chieh (1942) who found that a
complete cover of Azolla could prevent completion of mosquitoes life-cycle.
However, he pointed out that an incomplete mat provides protection to mosquito
larvae from their natural predators Germans successfully used Azolla for mosquito
control in the Rhine (Howard, 1910). The dense surface mat of Azolla was found to
interfere with oviposition of mosquitoes and also prevent adult emergence
(Amarsinghe and Kulasooriya, 1986) Extensive studies carried out in China to
evaluate the efficacy ofAzolla as a bio-control agent against mosquitoes in rice fields
which are important breeding habitats for Cx. tritaeniorhynchus, principal vector of
Japanese B. Encephalitis, showed that the complete coverage of A. filiculoides has
prevented oviposition of Culicine female mosquitoes (Luo Bao-Lin, 1988). The
inhibiting effect of oviposition was directly proportional to the coverage of aquatic
fern on water surface. Similar results were obtained with A. caroliniana against An.
sinensis a main vector of malaria. The complete coverage ofA. filiculiodes was also
detrimental to adult emergence in the case ofAn. sinensis and the average cumulative
reduction was 84.7 to 98.4% against larvae and pupae respectively. Indian scientists
have also reported that the complete coverage of A. pinnata significantly reduced
the hatchability of An. culicifacies eggs (Rajendran and Reuben, 1988). They have
also observed pupal mortality in the case ofAn. culicifacies and Cx. quinquefasciatus
in containers completely covered with A. pinnata.
Field evaluation carried out with Azolla microphylla (Rajendran and Reuben,
1991), where An. suhpictiis, Cx. pseudovishnui and Cx. tritaeniorhynchus breed in
rice fields predominantly indicate reduction of immature population after 13-14
days when 80% mat of Azolla was formed on water surface. When the density of
immature stages were compared for the overall study period (13 weeks) an appre
ciable reduction has been achieved in the larval and pupal density ofAnopheline and
('ulicine mosquitoes
Another field observation indicate considerable reduction in mosquito
breeding in habitats completely covered with Azolla (Ansari and Sharma, 1991). The
suppression of breeding was more pronounced against anopheline as compared to
cuhcine immatures. The percent reduction in anopheline immatuCe density was 93.6
f° °°o’"-io/00 S’ We"S aUd Paddy f‘elds’in comP,etely covered habitats as against
t
1
Pau y C?Vered habitatS- The Percent reduction
C/rto was
" '
3'7/ohabltats whlch were found completely covered with Azolla and in
4
partially covered habitats the percent reduction was in the range of 27.5 to 88 5% in
pools, wells, paddy fields, ponds and drains.
Discussion and Conclusion
When Azolla is inoculated to rice fields 7 days after transplantation, as
recommended at present, it takes about 5 weeks to multiply, grow and cover the
water body fully, whereas the intensity of mosquito breeding is very high during this
period. Thus partial coverage of water body by Azolla leads to poor control of
mosquito breeding, i.e , neither prevention of egg laying by wild mosquitoes nor
prevention of adult emergence. If Azolla inoculation is done just after transplanta
tion of rice seedlings, perhaps there is a possibility of faster coverage of the water
body by the fern leading to more effective control of mosquito breeding. Further,
enhancing the present application rate of Azolla (500 kg/ha) to 2000 kg/ha may also
lead to similar situation. But, logistical problems involved in transportation and
broadcasting of the large biomass need to be solved for a large scale control
programme
Azolla can multiply and grow faster during monsoon and post-monsoon
periods in tropical situations and incidentally rice cultivation as well as mosquito
breeding are intense during post-monsoon period. If the inoculation time and the
inoculation dose of Azolla are optimised there is better chance for it to be used in
the control of rice field breeding mosquitoes, especially, the vectors of IE virus In
situ production of Azolla along with rice nursery is advisable for inoculation to main
field, as this would avoid the problems associated with transportation of inoculum
produced elsewhere.
There is little information on the tolerance of Azolla to different pollutants,
especially organics, although some information is available on salinity tolerance and
data on these need to be generated. The problem of abundance of snails in Azolla
grown fields was highlighted. Simultaneous application of neem with Azolla did
not help much to prevent mosquito breeding.
5
Recommendations
• 1. More information should be generated on the potential of Azolla
for use in mosquito control (Dr. S. Kannaiyan, TNAU and Dr. N.
Balakrishnan, NICD).
• 2. Different Azolla species should be tested fortrapping nutrients and
heavy metals in polluted water bodies (VCRC).
• 3. Extracts of Azolla spp should be evaluated for mosquito control
potential (VCRC).
• 4. The alga, Westellopsis sp. should be tested for mosquito control
potential by extracting bioactive components. And a research project
proposal should be formulated along these lines for seeking funds from
DBT (CRME).
• 5. Inoculum level of Azolla for effective mosquito control should be
standardized (Dr. S. Kannaiyan, TNAU and Dr. N. Balakrishnan,
NICD).
• 6 The oviposition behaviour of mosquitoes in Azolla inoculated rice
fields and the detrimental effect(s) of Azolla mat on mosquitoes need
to be studied (Dr. S. Kannaiyan, TNAU and Dr. N. Balakrishnan,
NICD).
• 7. A common protocol for VCRC, CRME, TNAU & MRC, to be
drawn for studies on Azolla in rice fields.
List of participants.
1. Dr. S. Kannaiyan, Dean, TNAU, Coimbatore
2. Dr. N. Balakrishnan, Dy. A.D., NICD., Coonoor
3. Dr. M.A. Ansari, Dy. Director, MRC, Delhi
4. Dr. A. Gajanana, QIC, CRME, Madurai
5. Dr. R. Rajendran, SRO, CRME, Madurai
6. Dr. D. Raghunatha Rao, RO, CRME, Madurai
7. Dr. G. Rajendran, SRO, VCRC, Pondicherry
8. Dr. P.K. Das, Director, VCRC, Pondicherry.
6
Title of papers presented
1 Studies on the use of Azolla sp. for the control of rice field breeding mosquitoes.
Rajendran, R., Raghunatha Rao, D., and Gajanana, A.
2. Utility of Azolla in controlling of mosquito breeding in rice field. Ansari, M.A
j. Giowth and multiplication of the waterfern Azolla microphylla in wetland
rice ecosystem and its role in mosquito breeding, Kannaiyan, S.
4. Studies on the role of Azolla in controlling rice field breeding mosquitoes in
Coimbatore. Balakrishnan, N.
5 Feasibility of utilization of Azolla for mosquito control. Rajendran, G., and Jambulin
gam, P
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7
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8
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