70
Paper towel technique
Field survey around Mysore region revealed high incidence of sorghum downy
mildew, especially in local fodder ciltivars. To obtain the conidial inoculum of the
pathogen for use in experiments, diseased leaves need to be collected from fields in
the evening. The pathogen being a biotroph, cannot be cultured in vitro to obtain
the conidial inoculum. The method commonly followed to obtain the inoculum
involves plating of diseased leaves pieces in petri plates and incubating overnight in
incubator at 20ºC (Narayana et al. 1995; Pande et al., 1997). Numerous shortcomings
of this popular technique necessitated the development of an alternative simpler
technique termed as paper towel technique. Comparative evaluation of Petri-plate
incubation and paper towel technique was carried out.
Although Petri plate method showed highest sporulation, many advantages
associated with paper-towel method made us to adopt this method for further
experiments. Firstly, in the Petri plate incubation method, cleaning the leaves with
moist cotton, cutting into pieces, lining the petri plates with filter paper, plating and
harvesting the leaf pieces is laborious and time consuming. Secondly, the number of
9 cm diameter Petri plates required to plate 100 sorghum leaves would be at least
400-500. Even if sporulation in Petri plates is double than that of paper-towel, the
need of 200-250 plates makes the experiment formidable. In contrast, the cost of
paper-towel is much lower and each one can generally pack 8-15 sorghum leaves.
Thirdly, in the morning during conidial harvest, when speed of execution is crucial
because of ephemeral nature of conidia, the washing of full length leaves obtained
from paper-towel is faster than washing of leaf pieces obtained from petri plates.
Fourthly, the spores which cause disease transmission in nature are the ones which
are liberated in the air. In paper-towel method, the liberated spores are trapped as
conspicuous thick deposits on paper-towel and can be brushed off easily. Last but
not the least, the conidial inoculum of obligate parasite cannot be obtained from
cultures and is precious to researchers working in labs without facility of an
associated sick-field, as it is to be collected from far-off farmer‘s fields, which makes
early morning field visits difficult. Since in paper-towel method, the leave‘s cutends are dipped in water, they have longer viability lasting up to a week in
favorable climate and can be repeatedly used to obtain inoculum in consecutive
experiments day after day. In Petri plate incubation, leaf-pieces get dehydrated after
71
single use. The virulence of spores produced from the leaves collected 2 days and 4
days in advance, seems to increase surprisingly, rather than decreasing, as
compared to standard graph obtained from day 1 inoculation. Conidia were also
found to be more robust under microscope on day 4 as compared to day 1 and day
2. This crucial observation needs further investigation. Experience has shown that
covering of paper-towel-packed or even openly placed leaves (bouquet), with
polythene bag during overnight incubation gives better results. Although the
present experiment was conducted in May-June, we have used the paper-towel and
bouquet methods as reliable methods to obtain conidial inoculum throughout the
calendar year for other regular experiments and thus we feel confident that the
method can produce satisfactory results in early morning temperature range of 16.1
ºC to 21.4 ºC. The paper towel method may be used with incubator to overcome the
drawback of low sporulation.
Comparative evaluation of composts and uncomposted biomass:
Comparative evaluation of composts and uncomposted dry powders was
carried out at 4% (w/w) rate. As composts, 4 botanicals suppressed the disease,
while in dry powder form, 8 botanicals suppressed the disease. Besides, 2 dry
powders were found to promote the disease. Mechanism of action of botanicals is
thus not via release of nitrogenous compounds or organic acids which are exuded
by all organic and inorganic fertilizers upon decomposition (Tenuta and Lazarovits,
2002). Mechanism is thus inferred to be through general antimicrobial action of
secondary metabolites which bring down the total fungal count of soil. While the
toxicity for microbes persists longer in case of H. suaveolens, E. globulus and N.
oleander, the other three botanicals (A. cepa, L. camara, P. longifolia) are more ecofriendly and allow the micro-flora level to jump back to normal levels within seven
weeks of soil application.
All botanicals which suppress downy mildew of sorghum have proven track
record as general anti-microbials (Chanda et al., 2010; Dharmagadda et al., 2005;
Nantitanon et al., 2007; Rakhshandeh et al., 2004, Tagoe et al., 2011). Three botanicals
have been shown to reduce fungal population of soil in our studies. The possible
explanation for mild promotion of soil fungal population in case of three other
botanicals is that they lose toxicity by biodegradation at faster rate so that initially
72
when added to the soil they reduce the microbial population over first 3-4 weeks
but after losing the toxicity, the resultant non-toxic organic biomass acts as nutrient
source for microbial population which again rises over next couple of weeks.
Further studies need to be undertaken to study total fungal count after initial two
weeks and four weeks of botanical application.
There are numerous reports of control of soil borne disease by addition of
compost to the soil in green house and field conditions. Few earlier reports are
available about the use of dried organic powder for control of certain disease. Fritz
(2007) reported the control of pea root rot (Aphanomyces euteiches) by application of
rape seed powder to field furrows.
Besides, control of potato scab, potato
verticillium wilt and nematodes by application of high nitrogen containing organics
like soymeal, meat and bone meal was reported by Lazarovits et al (1999). Present
work is the first comprehensive screening of ten botanicals for their potential to
control soil borne propagules of a particular disease and it also involves
comparative evaluation of efficacy of composts versus dried powder.
Composting provides better nutrition but may cause simultaneous reduction in
disease control potential of botanicals, as is clearly evident from results obtained
with A. indica, E. globulus, T. indica and N. oleander, in our studies, all of which
suppressed the disease in dry powder form but promoted in compost form. Another
drawback in composting is difficulty of fine tuning of maturation time. Under-aged
and over-aged composts are neither effective for nutrition nor for disease control.
Perfect fine tuning of compost aging to get balance of nutrition and disease control
potential is desirable but difficult to achieve as it is dependent on multiple factors
like air temperature, physico-chemical and microbial properties of soil, pit size,
moisture content of material, pit aeration etc.
Dried powdered biomass has been found to be superior in many respects over
composts in management of the disease under study. Advantages associated with
powdered biomass are high shelf-life & low bulk facilitating storage & marketability
and also consistent composition & results. Thus success of present work and
methodology removes many difficulties associated with organic amendments.
There are few additional benefits of using dried organic powders over conventional
methods. Some botanicals like P. longifolia are initially toxic when added to the soil
but soon loose their toxicity by decomposition. Other botanicals like E. globulus
73
retain the toxicity for long time. Botanicals of former category may be added to the
soil a few weeks before sowing of crop so that they initially release antimicrobial
toxins, kill the pathogen and later loose toxicity by decomposition & provide good
source of composted biomass for the crop which would be sown few weeks later.
Quick loss of toxicity of organics is environment friendly & in sharp contrast to
chemicals used for soil sterilization which are soil pollutants, causing devastating
effect by bio-magnification. Seed treatment with systemic chemicals like metalaxyl,
besides posing risk of bio-magnification, protects the plant only while the pathogen
inoculum in surrounding soil is not affected and increases over the years.
Soil amendment with organics controls the disease by killing the pathogens in
the soil thus protecting the present crop & reducing the inoculum for the future
crops. Research methodology involved in soil amendment experiments is easier as
compared to resistant variety development, making it possible for farmers to test
different amendments for control of various diseases, reducing their dependence
and helping in self-reliance. The methodology has tremendous potential awaiting to
be tapped in future for control of all important soil borne diseases including wilts,
downy mildews of crops like pearl millet, maize, sunflower, grapes, to mention just
a few.
It is worth mentioning that the method is more suitable in case of obligate
pathogens (downy mildews) as the pathogen has no chance of multiplication in the
absence of host, once the propagule population is brought down by organics. In case
of saprophytes (wilts), the pathogen population can multiply back to previous or
higher levels by using organics as nutrient source (Lazarovits et al., 1999).
Screening of uncomposted botanical biomass
Comprehensive screening of 38 botanical amendments for management
oosporic infection showed that out of the six botanicals showing disease
suppressive potential, three (H. suaveolens, E. globulus and N. oleander) reduced
fungal population of soil, while other three botanicals (A. cepa, L. camara, P.
longifolia) promoted it significantly (Figure 1). Mechanism of action of botanicals is
thus not via release of toxic nitrogenous compounds or organic acids which are
exuded by all organic and inorganic fertilizers upon decomposition (Tenuta and
Lazarovits, 2002). Mechanism for 3 botanicals (H. suaveolens, E. globulus and N.
oleander) which reduce the total fungal count of soil is thus inferred to be through
74
general antimicrobial action of secondary metabolites, while the other three
botanicals (A. cepa, L. camara, P. longifolia) which enhance the soil fungal population
may act by influencing, antibiosis, competition or hyperparasitism by soil
microflora.
Screening of plant extracts
Plant products have shown promising results for control of soil borne
pathogens (Javaid and Saddique, 2012; Satish et al., 2009). Present work is the first
available report of comprehensive screening of 38 botanicals for their potential to
control a particular soil borne disease (Noble and Coventry, 2010).
Plant extracts have been earlier employed in spray form for management of
downy mildew of maize (Kamalakannan and Shanmugam, 2009), downy mildew of
grapes (Doagostin et al., 2010), downy mildew of cucumber and late blight of potato
(Wang et al., 2004). There are also a few reports of successful use of plant extracts as
antisporulants in lab as well as field conditions (Deepak et al. 2005; Kamalakannan
and Shanmugam, 2009). In the present investigation, an attempt was made to screen
and evaluate the anti-pathogenic potential of locally available plants against
Perenosclerospora sorghi, the causal organism of downy mildew of sorghum.
Although the methods employed in the experiment i.e. inoculum-antimicrobial
consortium assay and sprout inoculation are not akin to field conditions, yet the
technique proved effective in greenhouse screening of plant extracts for their
potential to suppress infectivity of conidia. The results reveal that at 20%
concentration of plant extracts, 8 plant extracts controlled the disease at par with
chemical fungicide to 0%. Datura metel extract also showed significant plant growth
promotion in terms of dry biomass yield (Fig. 25).
Formation of bulb like swellings in Petri plates treated with plant extracts and
chemical fungicide indicates the mode of action of plant extracts to be through
contact antimicrobial action via inhibition of germ tube growth.
In the present work, considering the principles of organic farming, only crude
water extracts of commonly available plants have been used. Large numbers of
plants were screened to compensate for slim possibility of finding an effective water
extract at low concentration. Plan and methods of the present investigation were
kept at simplest but effective level (avoiding the use of exotic plants and solvent
75
extracts etc.), so that the final product might be economically produced at farm
level. Efforts are required to select an effective adjuvant to be used with extracts so
that these can be tested in field conditions by spray method. Since the main problem
in controlling diseases in graminaceous crops concerns economic feasibility due to
high cost of chemicals and low commercial value of crop (Kenneth, 1981),
availability of costless potential organic fungicides, with option to choose according
to accessibility, would be of great help to farmers.
Evaluation of selected botanicals as extracts
At 10% concentration, 12 plant extracts were found to significantly suppress
the conidial infection, of which three (P. hysterophorus, D. repens, and O. latifolia)
were at par with chemical fungicide. Parthenium hysterophorus was earlier reported
as a remarkable anti-sporulant against downy mildew of pearl millet (Deepak et. al.,
2005). Additional advantage of inoculum antimicrobial consortium assay is that it
eliminates the role of adjuvants while evaluating fungicidal potential. Generally, the
efficacy of a fungicidal foliar spray is additive effect of efficacy of the active
ingredient and the adjuvants. Usually when a commercially available fungicide is
used as positive control to evaluate a plant extract for disease controlling potential
in form of spray, it necessitates the addition of an adjuvant to plant extract. Since in
most cases the adjuvants added to commercial fungicides are superior to the freely
available adjuvants which might be added to plant extracts, it places the plant
extracts at a position of disadvantage. The combined effect of active ingredient of a
fungicide product and the fine-tuned adjuvant may surpass the combined effect of
plant extract and freely available adjuvant, even if plant extract has better disease
controlling potential than active ingredient of fungicide. The consortium assay
exclusively compares the fungicidal potential of positive control and treatment by
eliminating the role of adjuvants while screening potential fungicides. This is
especially useful in case of obligate pathogens where in- vitro evaluation of antipathogenic potential of plant extracts is not possible by techniques such as poison
food. Nevertheless, since at the final stage of field application, the efficacy in the
form of spray only counts, evaluation of mix of plant extract and available adjuvant
becomes essential and was carried out successfully. High percentage of plant
extracts recording disease suppressive potential underlines the efficacy of new
technique as compared to spray method wherein many extracts with disease
76
suppressive potential may go unrecorded due to inefficacy of adjuvant. Although
the methods employed in the experiment i.e. inoculum-antimicrobial consortium
assay and sprout inoculation are not akin to field conditions, yet this novel technique
proved effective in greenhouse screening of plant extracts for their potential to
suppress infectivity of conidia. The technique has wide ranging applications and
may be adapted for all (especially in vivo) antimicrobial evaluation works. Reduction
in germination percentage of conidia and formation of bulb like swellings in petriplates treated with plant extracts, both indicate the mode of action of plant extracts
to be through contact antimicrobial action via inhibition of germ tube growth. In the
present investigation an interesting observation was made that in spite of the
removal sporulated plantlets from the pots in the morning between 7 am and 9 am,
still during evening reading more sporulated plantlets were spotted and removed
frequently. This observation is in contradiction to the earlier reports that
sporulation did not take place during day time between 6 am and 8 pm (Safeeulla,
1976; Frederiksen and Rosenow, 1967). Logical conjecture is that, the first
sporulation of infected plantlet does not follow the natural photoperiodic rhythm. P.
hysterophorus is known to cause allergic reactions in humans, so large scale spray of
P. hysterophorus crude extract is not advisable. O. latifolia being a small herb, may be
difficult to collect in bulk. Therefore, 10% water extract of D. repens may be suitable
for spray. Since with advancing age seedlings become less susceptible to infection
by air borne conidia (Kenneth, 1981; Singh and Garampalli, 2012b), a spray schedule
of one spray daily in evening for initial 5 days after germination can give almost
complete protection to crop from air borne inoculum. Since 750 litres of fungicide
solution is required to spray one hectare of a sorghum crop (Indofil® Mancozeb 75%
WP, manufacturer instructions) and 8 g of D. repens biomass yields about 80 mL of
10% extract, so 75 kg of fresh biomass of D. repens would yield enough organic
fungicide for spray of a hectare of crop. The same treatment may be tried for
management of downy mildew of maize which is a serious disease world over,
caused by the same pathogen. Even though D. repens is known to be toxic to
mammals, as the fungicide spray is required only a for few days after
germination for effective disease management, so the chances of retention of
toxic residue in plant at maturity are minimal due to withering away of initial
(sprayed) leaves, hundreds of fold of increase in volume of plant and a few
washes provided by rain, sorghum being a rain-fed crop. Still toxicity tests are
77
warranted before D. repens extract could be recommended as fungicide spray by
concerned authorities. Alternatively, the next best candidate plants such as Oxalis
latifolia may be used.
Oospore viability tests
The germination test is usually not relied upon for determining viability of
oospores of oomycetes members because uncertainty about dormancy factor
(Rebeiro et al., 1971). It cannot be applied especially in case of obligate pathogens
like P. sorghi. Very few oospores (˂1%) show germination in laboratory conditions
(Pratt, 1978). Pratt (1978) has expressed about doubts about higher percent
germination reported by other authors due to apparent ‗false germination‘ obtained
because of hyphal growth of parasitic chytrid fungi, from within the oospores.
Maximum germination rate of 20% was obtained by French and Schmitt (1980). The
tetrazolium bromide (MTT) staining and plasmolysis tests have been described for
determining viability of spores by many authors (Ribeiro, 1978; Boccas, 1981;
Sutherland and Cohen, 1983; Singh et al., 2004; Lumsden, 1980; Groves and Ristaino,
2000; Mc Carren et al., 2009). In living tissue tetrazolium is reduced to insoluble red
formazan by dehydrogenase enzymes. Since dead tissue lacks active dehydrogenase
enzymes it remains unstained (Kopooshian, 1968).
Shortcomings of both techniques are well known but plasmolysis method is
considered to be more reliable as it gives lesser false positive results (Etxeberria,
2011). The inconsistent and controversial results given by MTT staining technique
originate from various factors. The interpretation of the colours of stained
protoplasts varies from author to author. Most authors consider the red stained
oospores as viable, blue coloured as activated and black stained or unstained ones
as non-viable (Sutherland and Cohen, 1983; El-Hamalawi and Erwin, 1986; Jiang
and Erwin, 1990), whereas other authors have found red colour to be associated
with morphologically abnormal non-viable oospores (Boutet et al., 2010; Delcan and
Brasier, 2001). Moreover, making a distinction between a mildly stained oospore
and unstained one is as difficult as distinguishing between an over-stained and
black coloured oospore. Hence the results are dependent on personal assessment of
the observer.
Permeability of wall layers seems to depend on factors like age of oospores,
pre-trearment of oospores, storage conditions and it plays a vital role in the staining
78
of protoplast (Etxeberria et al., 2011). The colour taken up by oospores has also been
reported to be dependent on age of oospores (Dyer and Windels, 2003; Etxeberria et
al., 2011) and storage conditions (Jiang and Erwin, 1990; Etxeberria et al., 2011).
False positive results have been reported for both the techniques but in higher
proportions with MTT staining than with plasmolysis (Etxeberria, 2011). Sutherland
and Cohen (1983) reported 5% false positive results with MTT staining, in case of
sterilized oospores of Phytophthora megasperma, while Pittis and Shattock (1994)
reported upto 49% with P. infestans. The false positive results have been interpreted
to be due to production of an extracellular, non-enzymatic reducing agent by living
matter present outside the oospores, that enters oospores and reduces the tetrazolic
compound inside the inactivated sporangium (Williams, 1980) or because of red
staining of physically abnormal oospores (Delcan and Brasier, 2001; Boutet, et al.,
2010).
The reasons for false positive results in the present experiment may be two fold.
Firstly, it is difficult to conduct the whole experiment in sterilized conditions
because the pathogen is not culturable and oospores obtained from shredded leaves
cannot be sterilized without killing unpredictable proportion of spores in the
process. The experiment can be conducted at the most in relatively-sterilized
conditions by treating oospores with mild sterilizing agent like chlorine water/
sodium hypochlorite (Safeeulla, 1976). In such conditions any living matter present
in suspension outside the oospores can synthesize the reducing agent which may
then diffuse into the oospores and reduce tetrazolic compound inside dead oospore
to give false positive result. Secondly, because the cell wall of Peronosclerospora
members is known to be very thick, standard autoclaving under 121 °C, 15lb for 20
minutes may at best only kill a proportion of oospores, which results in presence of
viable plasmolysed oospores even in sterilized sample. The standardized
amendment added to the soil causes drastic reduction in disease incidence
signifying its ability to reduce the infectivity of oospores but the oospores recovered
from the soil treated with amendment show only marginal reduction in viability as
determined by plasmolysis test. It may be explained considering that the
amendment strongly affects the infectivity of oospores by acting on germ tube of
germinated oospores but has weak action on dormant ones. Moreover, a stained or
plasmolysed oospore represents only a respiring or intact oospore, and not its
79
capacity of germination or infection (Bowers, 1990). The blue staining of the
sterilized oospores has even been interpreted to be due to residual activity of dead
spores (Boutet et al., 2010).
Standardization of amendments and extracts.
The effective amendment rate for the management of sorghum downy mildew
caused by soil borne oospore has been brought down to 0.25% (E. globulus dry
powder), while most other reports could manage the soil borne disease at or above
10% amendment level (Nobel and Coventry, 2010) and there has been no available
report of disease management by amendments below 1% rate (Huang et al., 2005;
Ismail et al., 2012). It has been possible because of systematic stepwise screening,
evaluation and standardization of amendments. Thus the amendment rate has been
brought down to the realistic field rate of about 3 tonnes /acre.
The minimum effective concentration of D. repens and P. hysterophorus extract in
form of spray is 5%. P. hysterophorus spray cannot be recommended, being a potent
allergin. The D. repens aqueous may be used at 10% concentration (after due toxicity
tests) to overcome possible dilutions from dew or mild rain. The protection obtained
by spray of D. repens extract (10%) in green house conditions was 90.5% (Table 20).
An earlier report demonstrated the protection provided by induction of resistance
by dipping of sorghum seed in D. repens extract (2.5%) to be 50.9% in greenhouse
conditions (Manjunatha et al., 2013). By combining both organic methodologies,
86.6% protection was obtained in field conditions (Table 26). There are number of
reports claiming synergistic effect of plant extract mixtures resulting in improved
disease control potential as compared to individual plant extracts. A careful scrutiny
of literature revealed shortcomings in methodologies and conclusions of most such
reports. In some reseach papers only formulations have been tested against
standard chemical, without comparison with individual plant extracts, while
claiming superiority of formulations (Opraeke, 2007a; Opraeke, 2007b; Opraeke et al.
2006). Another report concludes the mixtures to be effective and recommends the
same, in spite of better performance of individual extracts shown in the results
section (Shanker and Uthamasamy, 2009). Earlier studies had shown that the
effective inhibitory concentration of different plant extracts against conidial
infection of sorghum by Peronosclerospora sorghi was – C. arietinum=20%, A.
indica=20%, E. globulus=20%, P. hysterophorus=10%, D. repens=10%. (Singh and
80
Garampalli, 2012; Singh and Garampalli, 2013b). So this study was undertaken with
the selected extracts to compare the anti-pathogenic potential of individual extracts
to the extract mixtures, by keeping the combined concentration of plant extracts in a
mixture equal to the effective inhibitory concentration of individual plant extracts. It
was aimed to find out if the mixtures could be more effective because of possible
synergistic effect, than the individual plant extracts. The results of comparative
evaluation of individual plant extracts and formulations proves that the
performance of mixtures is usually either in middle range between the performance
of individual constituent extracts or inferior to both individual extracts. In such a
situation it is better to opt for the individual extract for 2 reasons i.e. firstly because
of superior performance of at least one member of the mixture in individual form,
secondly because of simplicity involved in preparation of individual extracts. For
example E. globulus 20% extract is not only superior in disease suppression but also
simpler to prepare than E. globulus 10% + A. indica 10% extract. Lower disease
control potential of C. arietinum 20%, A. indica 20%, E. globulus 20% extracts obtained
in the present work as compared to earlier report (Singh and Garampalli, 2012) is
inferred to be due to higher concentration of conidial inoculum used in the present
work and variation in biochemical constitution of plants from season to season.
There is one report available which claims superiority of mixtures over individual
extracts, in spite of keeping the combined strength of extracts of the mixture equal
to the strength of individual extracts (De Britto et al., 2012). This report seems to be
an exception which is unlikely to be repeated. When mixtures are to be compared to
individual extracts and the total added strength of extracts of a mixture is to be kept
equal to the strength of individual extracts, it obviously causes dilution of
constituent extracts of a mixture which is responsible for its poor performance.
Performance of a mixture can be at best an average of the performance of
constituent extracts in individual form. In short, in the present study no synergism
was found in extract mixtures and no advantage observed in use of mixtures over
individual extracts.
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Chapter 4. Purification and characterization of active compound in disease
suppressive botanicals.
Terpenoids have basic unit of isoprene, a C5 molecule. They are classified as
monoterpenes, sesquiterpenes (C10 & C15), diterpenes (C20), triterpenes, sterols (C30)
and carotenoids (C40), depending on whether they contain 2 (C10), 3 (C15), 4 (C20), 6
(C30) or 8 (C40) of isoprene units. Triterpenoids are divided into 4 groups- true
triterpenes, steroids, saponins and cardiac glycosides (Harborne, 1984).
Plant sterols are essential components of the membranes of all eukaryotic
organisms and are responsible for membrane permeability (Piironen et al., 2000;
Pose et al., 2009). Phytosterols have attracted attention because of their cholesterol
lowering property and control of related ailments (Moreau et al., 2002). Their
properties like anti-atherosclerotic, anti-ulcer, anti-inflamatory, anti-oxidative and
ant-cancer activities have been subject of review articles (Delgado-Zamarrero et al.,
2009; Berger et al., 2004). Antibacterial and cytotoxic activities against cancer cells
has been reported by numerous studies (Salvador et al., 1997; Melo et al., 2004; De
Stefani et al., 2000a; De Stefani et al., 2000b; Qi et al., 2013; Sharma, 1993). Anticancer
effects of phytosterol have been reviewed by Woyengo et al. (2009). The phyosterols
have been reported to incorporated to RBC membranes and destabilise the
membrane integrity (Hac-Wydro et al., 2012). The incorporation of sterol and sterol
like molecule in to cell membrane and resultant disturbance of membrane integrity
has been proposed to be responsible for their anti-bacterial and anti-cancer activity.
While saponins present in plant extracts show antifungal activity against most of the
fungi by their action on ergosterols present in fungal membranes, the oomycetes is
known to be resistant to saponins due to absence of ergosterol in their membranes
(Arneson et al., 1967). Culturable oomycetes members can incorporate exogenous
sterols into their membranes when sterols are added to growth medium and show
increased sensitivity to saponins (Oslen, 1973). Saponins possess fungicidal (Nikkon
et al., 2008; Zhang et al., 1986), antiviral (Dourmashkin et al., 1962; Poehland et
al.,1987; Rattanathongkom et al., 2009; Zhao et al., 2008), properties. Mechanism of
action of saponins is via complex formation with membrane sterols, resulting in
pore formation and loss of membrane integrity (Dourmasnkin et al., 1962; Glauert et
al., 1962; Bangham and Horne, 1962). Duranta repens crude extracts and solvent
extracts have been reported to possess cytotoxic, anti-microbial, anti-malarial, anti-
82
viral and anti-oxidant properties. (Ahmed et al., 2009; Nagao et al., 2001; Shahat et
al., 2005; Abou Setta et al., 2007). Presence of alkaloids, tannins, steroids, saponins,
flavanoids, triterpenoids, iridoid glycosides, lamiides, acetosides has been recorded
in solvent fractions of the plant (Jayalaxmi et al., 2011; Takeda et al., 1995; Rimpler
and Timm, 1974; Hiradate et al., 1999; Anis et al., 2002; Salama et al., 1992;
Subramanian and Nair, 1972). The mechanism of action of D. repens crude extract
and active fraction appears to be 2 fold. The incorporation of sterols destabilize the
germ tube membrane, which may be followed up by the action of saponins present
in crude extract or active fraction or the soap solution (Tween-20) added as an
adjuvant or wetting agent. Plants rich in sterols and saponins need to be explored as
anti-microbial agents against plant pathogens of oomycetes group.