Summer Research Fellowship Programme of India's Science Academies 2017
Evaluation of acaricidal effect of the leaf extract of Vitex
negundo Linn. on the two-spotted spider mite pest,
Tetranychus urticae Koch (Acari: Tetranychidae)
Shamika Shenoy
Dr. D.Y. Patil Biotechnology and Bioinformatics Institute, Pune
Guided by
Dr. N. Ramani
Division of Acarology, Department of Zoology, University of Calicut
1. Introduction
Acarology, the study of acari (mites and ticks) has gained much global recognition because
of its vital role in imparting a general awareness on the impact of mites and ticks in varied
fields of interest to man and environment. Mites belong to the phylum arthropoda, subphylum
Chelicerata, class arachnida and subclass acari. Their size range between 300–500 µm and
they enjoy ubiquitous distribution in all major terrestrial and aquatic habitats. Unlike mites,
ticks are larger and exclusively blood-feeding parasites of warm blooded vertebrates
including humans. Mites are the most diverse and abundant of all arachnids but because of
their small size, they are rarely noticed. A good quantum of mites serves as serious
pests/parasites in soil, water, house dust, stored products, hair follicle, feathers of birds, fur
of animals and on the under surface of leaves. As potential vectors, many species of mites
and ticks transmit some of the most devastating and lethal disease agents like the bacteria,
viruses and pathogenic protozoa. Soil mites, especially the oribatids often do not cause any
damage to plants, in fact they are deemed to be beneficial to the decomposition process. Also,
mites associated with insects can be agriculturally important as they act as
parasites/predators. Under optimal conditions, mites develop rapidly and can increase in
population quickly over a short period. This hyper-diverse group of mites include varied
habits like predaceous, phytophagous, mycophagous, saprophagous, coprophagous,
necrophagous, necrophagous, phoretic and parasitic.
The agricultural significance of mites routes from their phytophagous habit which helped
them to conquer and successfully colonise hundreds of crop plants of varying economic
utility. In fact, mites are the only plant-feeding arachnids which attracted the attention of
agricultural people and became the objects of primary concern to growers/farmer community.
The major phytophagous groups of mites recognized so far include members of the families
Tetranychidae, Tuckerellidae, Tenuipalpidae, Bryobidae, Eriophyidae, Phytoptidae,
Diptilomyoptidae, Tarsonemidae etc. which induce diverse types of plant
abnormalities/injuries, ultimately leading to heavy yield loss. Specifically, mites belonging
to superfamilies Tetranychoidea (e.g. spider mites, false spider mites) and Eriophyoidea (gall
mites, erinose mites, bud mites, rust mites) colonize almost all kinds of agricultural crops,
vegetables, fruit crops, ornamental plants, medicinal plants, forest plants and even weeds.
Of these, the spider mites of the family Tetranychidae became a principal concern of
entomologists following World War II. Among the non-insect pests, several species of the
spider mite genus Tetranychus are distinguished as major pests, causing damage, often
leading to 7–48% of yield loss (Anonymous, 1996).
Spider mites are highly polyphagous and mainly feed on vegetables such as tomato, cucurbits,
capsicum, French bean, strawberry and ornamentals namely rose, carnation, cyclamen,
gerbera etc. Infestations by most spider mites are favoured by hot, dry weather and
prevalence of high humidity tends to depress their population. These mites characteristically
have a stylophore and styletiform chelicerae. Plants regulate water retention and transpiration
through leaves as they contain vast array of stomata which open and close according to
environmental conditions. Thrusting action in back and forth directions with lubrication from
the salivary glands of spider mites causes the chelicerae to penetrate to a depth of 100µm
(Jeppson et al., 1975) and extract cell contents and fluid, puncturing the host plant’s
protective system with tiny holes. The damaged leaves lose too much water and become
dehydrated. As a result, the plant’s ability to photosynthesize diminishes. Bronzing also
occurs because of damage to the mesophyll tissue during feeding.
The life cycle of Tetranychid mites is completed within 713 days but it varies between
species. The optimal temperature for development of most species of mites is approximately
in the range of 2530ºC. Hot and dry weather is most conducive for their growth. Extreme
temperatures and humidity cause the mites to enter into a diapause phase. They develop
through egg, larva, protonymph, deutonymph and adult stages. The nymphal and adult stages
are initiated during intervening period of inactivity called protochrysalis, deutochrysalis and
teliochrysalis. During these periods, the mites anchor to the host leaf or to its webbing and
the legs are bent upon themselves.
Egg: The female spider mites lay eggs on the under surface of leaves, attached to the fine
silken webbing. The eggs are usually spherical, translucent, pale yellow in colour and are
distinct from the predatory mite’s eggs which are white. One female may lay up to 20 eggs
per day and can live for 2 to 4 weeks, laying hundreds of eggs. This high reproductive
potential allows mites to adapt resistance to pesticides.
Larva: When the embryo is fully formed, it hatches by rupture of the egg case to release the
hexapod larva i.e. with three pairs of legs and which begins to feed on the host. The other
immature stages have eight legs. The larvae take in enough food to survive in resting stages
and their colour changes and two dark spots appear on the body.
Nymphs: The larva remains dormant for 24 h and undergoes quiescence and moults into the
first nymph. Between each stage, i.e. from protonymph to deutonymph, and from
deutonymph to adult, is a quiescent/inactive phase, during which the instar remains settled
on the leaf with its legs drawn apart. The metabolic rate of the instar decreases during this
inactive phase. The protonymph is usually a free and actively feeding instar. The deutonymph
is the second nymphal stage in the life cycle and may differ from the adult only in size and
the patterns of sclerotization. The nymphal stages are characterised by ovoid body and eight
Adult: After a short period of quiescence, the deutonymph finally moults into the adult. The
adult mite has four pairs of legs and its body is divided into an anterior gnathosoma and a
posterior idiosoma. Spider mites are sexually dimorphic, the females are comparatively larger
and oval while the males are smaller and with tapered hysterosoma. The genital structures
are clearly distinct in the males and females.
1.1. Chemical acaricides as tools to control pest mites
Attempts to control pest mites through the application of chemical acaricides were initiated
long back and in the 1920s sulphur was the only acaricide used to control mites. As the
members of Tetranychus and Panonychus showed resistance against sulphur, tar oil dormant
sprays were used subsequently to kill the eggs of aphids, the European red mite, P. ulmi, and
Bryobia spp. The tar oils were followed by dormant oils; then in the 1930s, dinitro
compounds were incorporated in the oils for ensuring more effective egg mortalities. The
development of dinitrophenol (DN) type acaricides, formed the first of the organic acaricides
which produced phytotoxic effects when applied during hot weather and were relatively
ineffective during cool weather. Residue persistence was short; therefore repeated treatments
often required. The surge of synthetic acaricide development began in the late 1940s. Nervous
system of mites has long been the target for most synthetic acaricides. Pesticides under
different groups i.e. organophosphates, carbamates, organochlorines, formamidines,
pyrethroids etc. were used to combat population of mites. Also, some tetronic acid derivatives
were used which imparted direct lipid synthesis inhibition in mites. Moreover, widespread
use of neuroactive insecticides, destroyed spider mite predators, generally more susceptible
than their preys; on the other hand, heavy selection pressure by neuroactive insecticides
caused emergence of tetranychid mite populations resistant to these compounds. Sprays
containing ammonium potassium selenosulfide and rotenone marked the first advance in
spider mite control in greenhouses. These sprays were highly successful at first, but soon
became ineffective. Resistance to Selocide was first encountered in 1937 and its use being
discontinued by 1947. Different stages of red spider mites were found susceptible to different
toxicants; therefore combinations of two or more acaricides came into use; especially in the
early stages of resistance development.
Chemical applications to control pests often become a major factor in upsetting the natural
balance; therefore knowledge of the effects of pesticide treatments on subsequent mite
infestations may aid in providing the most economical control of mite populations.
Bioaccumulation and enhanced persistence of acaricides in the food chain tends to increase
concentration of the chemical, leading to biomagnification. Several studies indicate that the
two-spotted spider mite populations that develop resistance to an acaricide by repeated
treatments may have become more susceptible to adverse conditions in the environment. This
may be reflected by lower egg production or viability, a longer life cycle, decreased ability
to survive weather extremes for exposed mites than for mite populations not having been
exposed to repeated treatment by the acaricide. Closely related species differ in their
physiological susceptibility to acaricide applications. An effective acaricide need to be toxic
to only one tetranychid mite stage providing residues are toxic throughout the life cycle of
the mite. Synthetic acaricides are non-specific and have an adverse impact on beneficial
insects and predators of mite pests. Also, adjuvants are added to the acaricide to improve its
performance, making it economically unprofitable.
In order to search an environmentally safe alternative, synthetic pesticides are replaced with
pesticides having biological origin. Many plant species produce substances that protect them
by killing or repelling the pests that feed on them. Natural pesticides have many advantages
over synthetic ones and may be more cost-effective as a whole, considering
the environmental cost of chemical alternatives. Natural pesticides are biodegradable, barely
leave residues in the soil and are less likely to harm humans or animals. They are usually
characterized by low mammalian toxicity, reduced impact on non-target organisms and short
persistence in the environment (George et al., 2008).
Due to its short life cycle, abundant progeny and arrhenotokous reproduction, T. urticae is
able to develop resistance to acaricides very rapidly. As a result, it is considered as one of the
“most resistant species” in terms of the total number of pesticides to which populations have
become resistant, and its control has become problematic in many areas worldwide.
On a global level, populations of T. urticae and Panonychus ulmi have been reported to
develop resistance to various acaricides since 1990s (Osakabe et al., 2009). It is astonishing
to record that among the 20 species of resistant agricultural and medical arthropod pests
tested, T. urticae was ranked as the most resistant one while P. ulmi occupied the 9th position
of the most resistant category (Whalon et al., 2008). It is high time that safer alternatives need
to be developed to suppress T. urticae through biological means, through mass rearing and
release of selected natural enemies or through the formulation and application plant derived
biopesticides. Taking into consideration of this, attention was focussed during the present
work to formulate different concentrations of the bioactive principles contained in the leaf
extract of a medicinal plant, Vitex negundo in different solvents and to evaluate the acaricidal
potential of individual concentration against the life stages of the pest mite, T. urticae.
2. Review of literature
The present review incorporates a concise report on the research work carried out for the last
few decades, on the effect of various acaricides on populations of phytophagous mites.
Considering the diverse types of control strategies practiced against the insect pests in general
and the mite pests in particular, the review of literature presented here has been arranged to
cover four major themes as discussed below.
Phytophagous mites have attracted the attention of acarologists and progressive farmers
owing to the diverse levels of interactions with their host plants which in most instances were
known to induce significant damage and yield loss (Jeppson, et al., 1975; Meyer, 1981; Keifer
et al., 1982; Gupta, 1985, 1991, Helle and Sabelis, 1985; Singh and Mukherjee, 1989, Wilson
et al., 1998; Walsh et al.1998; Singh et al., 2000). A total of 660 mite species were known to
infest on various plants of India, out of which 319 were found to represent the
phytophagous/predatory category (Gupta, 1975, 1985, 1991; Gupta and Nahar, 1981; Nassar
and Ghai, 1981; Singh et al., 1987; Singh and Mukherjee, 1989; Yadav et al., 2001). The
plant feeding mites were found to include 169 species belonging to Tetranychidae (>69
species), Tenuipalpidae (>29 species), Eriophyidae (>64 species) and Tarsonemidae (>4
species). Of these, 17 species were reported as major pests on various plants.
Chemical control of mites and other plant pests: With reference to past events, the oldest
chemical treatment used against mites was sulfur (Hubbard, 1885). Later, petroleum oils were
used as acaricides. Various chemical acaricides were introduced that targeted different
systems of plant pests. Various chemical pesticides like the organophosphates, carbamates,
organochlorines, formamidines, pyrethroids and avermectines were found affecting the
nervous system of mites (Dekeyser and Downer, 1994; Knowles 1997). Some acaricides
inhibited the respiration. Mitochondrial Electron Transport Complex- I (MET- I) acaricides
fenpyroximate (pyrazole-oxime), tebufenpyrad (pyrazole-carboxamide), pyridaben
(pyridazinone), and fenazaquin (quinazoline) gained popularity worldwide and were used for
insect management (Sparks and DeAmicis, 2007; van Leeuwen et al., 2010). These
compounds were reported as effective against spider mites and eriophyoid mites, as well as
against hemipteran and lepidopteran insect pests (Dekeyser, 2005; Sparks and DeAmicis,
2007). MET-II complex inhibitors were beta-ketonitrile derivatives cyenopyrafen and
cyflumetofen acaricides and which were intended for spider mite control (Tomlin, 2009).
Development of synthetic acaricides: The advent of synthetic organic insecticides in the
mid twentieth century made the control of insects and other arthropod pests much more
effective, and such chemicals were listed as essential in modern agriculture despite their
environmental drawbacks. Nervous system of mites was the target for most of the chemicals
which were used for their control (Casida and Quistad, 1998). Such synthetic organic
compounds included benzoyl acetonitriles, diphenyls, carbamates, carbazates and
dinitrophenols and many more. Spider mites were reported to have developed resistance
against diphenyl carbinols (Cranham and Helle, 1985) and hence their use was banned.
Organochlorine insecticides such as endosulfan and DDT and acricides like dienchlor were
found to have little effect on tetranychid mites (Childers et al., 1996). In general, carbamates
were much more effective against eriophyoid mites than the tetranychids (Childers et al.,
1996) and it was reported that carbamates like bifenazate, were effective against all stages of
the two spotted spider mite, T. urticae (Ochiai et al., 2007).
Pest resistance to the chemicals and other disadvantages: During the last two decades,
due to indiscriminate spraying of broad spectrum pesticides for control of agricultural pests,
many of the mite pests which were either innocuous or of very little importance in the past
were reported to have assumed the status of major pests, not only in India but in many
advanced countries as well. Besides, due to repeated use of organophosphoric compounds
and many a times at their sublethal doses, plant mites developed resistance/cross resistance,
which made their control a more difficult task. The development of insecticide resistance was
found influenced by many factors, including genetics, biology/ecology and control operations
(Georghiou and Taylor, 1977). There were many possible adaptations that would permit an
insect or mite to survive lethal doses of an insecticide/acaricide. These were usually classified
based on their biochemical/physiological properties, as either mechanisms of decreased
response to the pesticides (interaction of a pesticide with its target site), or mechanisms of
decreased exposure (penetration, distribution, metabolism and excretion) (Roush and
Tabashnik, 1990; Feyereisen, 1995; Taylor and Feyereisen, 1996).
The most recent trend followed in resistance research included giving major emphasis to
unravel the underlying molecular mechanisms, and to apply this knowledge to control the
development and spread of resistant populations. A large number of compounds with different
chemical structure and mode of action were identified, including neurotoxic insecticides such
as organophosphates and pyrethroids, specific acaricides such as Mitochondrial Electron
Transport Inhibitors (METI’s) and organotins, and recently developed compounds such as
ketoenols (Knowles, 1997; Dekeyser, 2005; Van Leeuwen et al., 2009). T. urticae was
distinguished as a notorious species which could rapidly develop resistance to chemicals
(Knowles, 1997; Van Leeuwen et al., 2008). Development of resistance was found accelerated
by a combination of features possessed by the species such as the high fecundity, inbreeding,
arrhenotokous reproduction and very short life cycle resulting in many annual generations,
especially during warmer conditions (Cranham and Helle, 1985; Van Leeuwen et al., 2009).
Based on these features, T. urticae could be assigned as ‘most resistant’, in terms of the total
number of pesticides to which populations developed resistance. Recently, molecular
characterisation of the various resistance mechanisms in T. urticae was also done at different
levels (Van Leeuwen et al., 2008; Tsagkarakou et al., 2009; Khajehali et al., 2010).
Evaluation of the efficacy of botanicals in the control of phytophagous mites: The use of
chemical acaricides was proved increasingly inefficient and caused various problems of
ecological concern As a result, attempts were initiated to suppress plant damage by spider mite
pests and other plant pests through the development of bioacaricides.
Patel et al. (1993) pointed out that excessive reliance on chemicals would lead to an imbalance
in natural regulatory factors and this would affect the yield of mite infested crops, leading to a
yield loss of 30 to 40%. This necessitated the need for using perishable goods with safer and
biodegradable products, thereby favouring the formulation of biopesticides. As a result,
biopesticide research became a fascinated area among the scientific sector and probably the
most studied botanical insecticide during the last 20 years would be the triterpenoid,
azadirachtin. Results of studies made on plant-feeding mites (Mansour et al., 1997; Martinez-
Villar et al., 2005; Venzonetal, 2008), proved the acaricidal efficacy of azadirachtin, by
inducing mortality, repellency, and reduction of fecundity and longevity in pest mites.
Copping and Duke (2007) briefly described the importance of using compounds as starting
materials from living organisms in crop protection for semi-synthetic derivatives. Kumaran et
al. (2007) investigated the bioefficacy of botanicals against the two-spotted spider mite,
T.urticae infesting Okra and the results of the trials revealed that Azadirachtin (1%) was the
most effective formulation against T. urticae, registering population reduction of 70.16 to
70.95% followed by TNAU Neem oil ‘A’ 60 EC (49.21 and 43.90) and TNAU Neem oil NO
‘C’ EC (65.83 and 46.90) over the untreated check, respectively in the first and second season,
Investigations carried out by Manjunatha Reddy et al. (2009) to evaluate the effect of 5 and
10 per cent concentrations of the aqueous extracts of leaves and barks of Cinnamomum and
Jatropha against T.urticae. These extracts were assessed for repellence, mortality and
oviposition deterrence of T. urticae. Statistically, leaf extracts of C. camphora at 10 per cent
concentration showed highest repellency, while the bark extracts at 10 per cent concentration
imparted maximum mortality.
Chandler et al. (2011) discussed the challenges and opportunities for Integrated Pest
Management (IPM) in the developed economies, with emphasis on the European Union (EU).
The authors studied the development, regulation and use of biopesticides for IPM emphasized
on different types of biopesticides according to the active substances based on living
microorganisms, biochemical and their commercial utilization. Afify et al. (2011) used six
concentrations of extracts of Syzygium cumini (Pomposia) at concentrations of 75, 150 and
300 g/mL to control T. urticae and observed that the ethanol extract had the maximum
acaricidal activity leading to 98.5% mortality followed by hexane extract (94.0%), ether and
ethyl acetate extract (90.0%). Roh et al. (2011) screened the acaricidal and oviposition
deterrent activities of essential oils of 34 species of plants on the pest mite, T. urticae and the
results of the study confirmed that only sandalwood oil had significant effect, causing 87.2 ±
2.9% mortality.
By adopting adhesive tape and residual film methods, Yanar et al. (2011) tested the potential
of 12 plant species as acaricides against T. urticae and found that the methanol extracts of
Lolium perenne (flower, leaf), Anthemis vulgaris (flower) and Chenopodium album (flower,
leaf) induced significantly higher rates of mortality than that of azadirachtin (10 g/L) and
synthetic pesticides. Studies were carried out by Attia et al. (2011) to examine the acaricidal
properties of essential oils of Deverra scoparia against the serious pest T. urticae. It was
observed that female mortality increased with increasing D. scoparia oil concentrations, with
LD50 and LD90 values at 1.79 and 3.2 mg/l, respectively. Attla et al. (2011) conducted field
experiments with the extracts of seven plant species (Haplophyllum tuberculatum, Deverra
scoparia, Mentha pulegium, Chrysanthemum coronarium, Hertia cheirifolia, Citrus
aurantium and Santolina africana) and results of which indicated that these extracts were
effective leading to reduction in the population density of T. urticae at 0.30, 0.36, 0.37, 0.46,
0.48, 0.50 and 0.53 mites per leaf respectively.
Gonzalez-Coloma et al. (2013) explored the natural based biopesticides for the control of
insects, which revealed the potential of various compounds such as Karanjin from pongam
which could be used for control of mites, scale insects, chewing and sucking pests and some
fungal diseases. Nicotine, the main bioactive component of tobacco plant could be very
effectively used for the control of insects, aphids, thrips, whitefly and many more. Syahputra
and Endartob (2013) studied the acaricidal activity of various tropical plants against the citrus
rust mite, Phyllocoptruta oleivora and the citrus red mite, P. citri and found that the extract
of J. curcas exhibited the strong acaricidal activity against P. oleivora with LC
of 0.8%.
Alawi (2014) evaluated the acaricidal activity of extracts of Ruta chalepensis, Astragalus
oocephalus and Urtica pilulifera which showed that for all the extracts, mortality rate
exceeded 50% against developmental stages of T. urticae. Five acaricides, Challenger, Ortus,
Vertimec, Delmite and Bioca were investigated for their efficacy in controlling the pest mite,
T.urticae by Abou El-Ela (2014) and the authors also tested their side effects on the natura;
enemies like predatory insects, mites and spiders. Of these, two of acaricides, Challenger
and Ortus were markedly more efficient in reducing the mite population than Vertimec,
Delmite and Bioca as they caused 81.55% and 80.62% reduction in average within 3, 7, 14
and 21 days after spraying at early season, respectively.
Satilal Bhika (2014) sprayed various chemicals like Kelthane, Vertimec, Magister, Omite,
Missile and M-Impact in specified concentrations against the pest mite, T. urticae infesting
rose plantation and recorded that chemical miticides though reduced mite density, the
botanical miticide i.e. M-impact showed the highest mortality on the two spotted spider mite.
Lakhdari et al. (2015) conducted experiments to study the effect of aqueous extracts of
Zygophyllum album, Cotula cinerea and Limoniatrum guyonianum on the mortality of the
date palm mite, Oligonychus afrasiaticus and found that these plant extracts except Z. album