Phenolic Acids Associated with Sclerotium rolfsii in Groundnut
(Arachis hypogaea L.) During Pathogenesis
Stem rot caused by Sclerotium rolfsii is a potential threat to groundnut
yield. This disease causes severe damage during any stage of crop growth and yield
losses over 25%. Isolation trails from diseased plants were carried out in some
areas of Srikalahasti and Tirupati of Chittoor district. Oatmeal-sand culture
of S. rolfsii was inoculated to 10 day old seedlings of TMV-2 variety
of groundnut for disease development and divided into five fairly distinct stages,
on the basis of lesion development. Phenolic extracts were prepared from the hypocotyl
regions with lesions and healthy seedlings respectively. The extracts were subjected
to 2-dimensional ascending paper chromatographic technique by employing the benzene:
acetic acid: water as first solvent system and sodium formate:formic acid:water
as the second solvent system. There is a tremendous increase in qualitative and
quantitative production of phenolic acids in the diseased area of the groundnut
hypocotyls compared to healthy ones. Chromatographic data revealed that there
are 10 phenolic acids in healthy plants, whereas in the infected plants, in addition
to the above 10 phenolic acids, 6 additional compounds were detected with the
progression of the disease.
Received: November 05, 2011;
Accepted: March 21, 2012;
Published: June 05, 2012
Sclerotium rolfsii is a ubiquitous soil-borne fungal pathogen known
to cause disease on worldwide range of agricultural and horticultural crops
(Kaveriappa, 1979; Sennoi et
al., 2010). Among plantae, more than 500 species belonging to 100 families
are susceptible to this pathogen (Punja, 1985; Sarma
et al., 2002; Adandonon et al., 2005;
Pandey et al., 2005; Ganesan
et al., 2007). It has been reported that most of S. rolfsii
infections were identified in dicots and also in some monocots (Aycock,
1966). Patil and Rane (1983) reported that all the
35 hosts, including important cultivated crop plants, were susceptible to the
pathogen, indicating the wide host range of parasitism of S. rolfsii.
Numerous secondary hosts were identified to this pathogen, among which food
crops and ornamental plants that are economically important are included. Secondary
hosts also include a variety of hosts.
S. rolfsii devastates the plant severely at any stage of crop its growth
and is detrimental to all parts of the plant, but stem damage is the most common
and serious risk (Ganesan et al., 2007). As a
protective structure, sclerotia possess viable hyphae which serves a primary
inoculums for disease development (Aycock, 1966; Nalim
et al., 1995; Cilliers et al., 2000)
as well as its principle means of dispersal and sole organs by which the fungus
survives adverse environmental condition, germinates and infects the susceptible
hosts during favourable conditions. The disease manifests itself in the form
of dark sunken necrotic lesions girdling the hypocotyls near soil level. The
necrotic lesions formed due to infection with S. rolfsii are attributed
by many to be due to some diffusible phytotoxic metabolites or toxins (or more
precisely the pathogen-produced determinants of disease) produced
by the fungus (Kerr, 1956; Wyllie,
1962; Sherwood and Lindberg, 1962; Aoki
et al., 1963). The present study was undertaken to evaluate quantitative
changes in the phenolics of groundnut hypocotyls brought about by S. rolfsii
infection and to determine the nature of phenolic compounds produced by
the pathogen in vitro.
MATERIALS AND METHODS
Isolation of pathogen: During the survey of groundnut fields around Srikalahasti and Tirupati areas of Chittoor District and also other Rayalaseema Districts of A.P., Sclerotium rolfsii was found to be associated with the infected hypocotyls region of groundnut variety TMV-2 at early stages of growth and development. The plants showing stem rot or southern blight symptoms were brought to the laboratory for making isolation according to the tissue segment method on PDA, pure culture was obtained by transferring the sclerotia to PDA plates. The stock culture was maintained on PDA slants in a refrigerator and subcultured for every two months.
Method of raising plants: High quality seed material (average 95% germination) of groundnut variety TMV-2 was obtained from the local Agricultural Research. Sound seeds were surface sterilized with 0.1% mercuric chloride for 2-3 min followed by repeated washings with sterile water and sown in sterilized soil contained in seed pans. The seeds germinated and emerged in 4-5 days. One week old seedlings were used for inoculation purpose.
Inoculation: Ten days old oatmeal-sand culture of S. rolfsii was thoroughly mixed with sterilized soil at 10%. This inoculum-soil mixture was then distributed in 12" diameter earthenware pots and left undistributed for two days. After this period, one week old seedlings grown in seed pans were lifted carefully without causing much damage to the root system and transplanted into the pots. They were watered on alternate days and kept in an open atmosphere.
Disease indexing: The plants were periodically examined for the progress
of the disease. Samples were collected at random from four pots each time at
0 h, 2 days, 5 days, 9 days and 11 days after inoculation. Almost
all the seedlings collapsed by 11 days after transplantation. The progress of
disease in the hypocotyls of the seedlings could be differentiated into the
following five fairly distinct stages, on the basis of lesion development:
||0 hours, i.e. immediately after inoculation: Healthy seedlings
|| 2 days after inoculation: The early or young phase, characterized by
water-soaked appearance of invaded portions of hypocotyls which remained
almost colourless or were light brown in colour
||5 days after inoculation: The intermediate stage, in which the lesion
surface become brown to dark brown colour
||9 days after inoculation: Well developed, dark necrotic lesions often
girdling hypocotyls. This marks the final stage in lesion maturation. The
lesions also showed sunken appearance
||11 days after inoculation: Characterized by dry appearance of the lesion
surface. Downward destruction or rotting of the tap root occurred and then
the seedlings wilted and died
Symptoms characteristic of each of the above stages of lesion maturation are shown in Fig. 1.
Collection of host tissue: For extraction of phenolics, hypocotyl regions with lesions and the corresponding hypocotyl portion of healthy seedlings were collected at the five different stages of lesion development cited above. They were washed thoroughly with distilled water to remove adhering soil particles and used immediately for extraction.
Changes in phenolic acids: Phenolic acids were extracted according to
the method of Bate-Smith (1954) adopted by Das
and Rao (1964). About 10-15 g of fresh material was extracted in 2 N HCl
and digested on a boiling water bath for 20 min. The digest was filtered and
the filtrate shaken with diethyl ether to extract phenolic acids into it. It
was heated for 20 min and then shaken several times with ether (Reddy
et al., 1975). All the other extracts were combined and the phenolic
acids were taken up from the combined extract into 5% sodium carbonate which
was acidified and reextracted with ether. The final ether extract was subjected
to paper chromatographic analysis using a 2-dimensional ascending technique
on Whatman No.1 chromatography paper. The solvents employed were benzene-acetic
acid-water (60:70:30 upper phase) in the first direction and the sodium formate-formic
acid-water (10:1:200) in the second direction (Ibrahim and
Towers, 1960). Papers were run twice in the same direction in the first
solvent to ensure better resolution.
The dried chromatograms were observed under ultraviolet light, first without
and then with ammonia vapors, all the fluorescent spots were marked. The sheets
were then sprayed with diazotized p-nitraniline (Smith, 1960)
or diazotized sulphanilic acid (Ames and Mitchell, 1952)
or 1% ferric chloride in order to identify the phenolic acids present in the
extracts. Authentic samples were also developed under identical conditions.
||Groundnut seedlings inoculated with S. rolfsii, 1:
Immediately after inoculation, 2, 3, 4 and 5: Disease development on stems
until the maturation of lesions
Identity of some of the compounds was also confirmed by co-chromatography
with authentic samples. The areas form unsprayed sheets, corresponding in Rf
values to the detected phenolic acids, were eluted with 90% ethanol. The eluates
were evaporated to dryness and dissolved in 3 mL of distilled water; 0.5 mL
of Folins reagent was added and thoroughly shaken. After 3 min, 1 mL of
saturated sodium carbonate solution was added and the mixture was made up to
10 mL. After 1 h, the blue colour developed was read at 725 nm using Spectronic-20
colorimeter. The phenolic acid content of the individual spots was calculated
by making use of the standard curves prepared from their respective authentic
samples. The quantities of unidentified compounds were calculated from the standard
curves prepared with known quantities of chlorogenic acid.
Phenolic acids occurring in healthy and S. rolfsii infected groundnut hypocotyls are presented in the Table 1. There is a tremendous increase in the number of phenolic acids in the diseased area of the groundnut hypocotyls. Chromatographic data revealed that there are altogether 10 phenolic acids in healthy plants, nine of which were identified as trans-caffeic acid, chlorogenic acid, cis-caffeic acid, cis-p- coumaric acid, p-hydroxybenzoic acid, trans-p-coumaric acid, trans-ferulic acid and vanillic acid and remaining one was unidentified. Infection resulted in a tremendous effect on the qualitative nature of phenolic compounds resulting also in the production of new or additional compounds. In the infected plants, in addition to the above mentioned ten phenolic compounds 6 additional compounds were detected with the progress of disease.
||Effect of S. rolfsii infection on phenolic acid content
(mg g-1 fresh weight) of healthy and infected hypocotyls at various
stages of disease development
|Each value is an average of 3 replicate samples
Many fungi produce metabolites in culture which are phototoxic and it is possible
that such substances are released by fungi in the course of growth in their
host tissues and they are involved in the production of disease symptoms (Reddy
et al., 1975; Basha et al., 2006).
The point of interest in this investigation is that significantly greater accumulation
of phenolic compounds is observed in diseased plants than in healthy ones and
that a correlation exists between the compounds produced by the pathogen in
vitro and some of those that appeared a fresh in diseased plants. It is
interesting to note that salicylic acid which also reported to induce resistance
in host plants against pathogen attack was not detected in sclerotial exudates
of Sclerotinia sclerotiorum (Basha et al.,
2006). The occurrence of the new compounds in the S. rolfsii infected
tissue may be due to the production by the fungus because the corresponding
similar compounds were also observed in the culture filtrate. The production
of several new compounds in the infected tissues indicates that they are apparently
formed through the interaction of the fungus and the living host since no such
compounds were observed in extracts of the healthy host and also neither in
the fungus nor in culture filtrate of the fungus. It is also well established
that the processes and mechanisms associated with disease development are a
junction of both the host and the pathogen and that the disease determined abnormal
physiological activity, strictly speaking, may not be characteristic of either
the host or the pathogen individually but of the host-pathogen complex (Bateman,
1970). The fluorescent compounds may be involved in localization of the
fungus and may limit lesion size (Deverall and Wood, 1961).
The chromatographic data, precipitation with lead acetate, fluorescence in UV radiation and reaction to different spray reagents used suggest that the fluorescent compounds are phenolic and acidic substances. Negative reactions of several unidentified compounds with diazotized sulphanilic acid, diazotized p-nitraniline and ferric chloride do not support this conclusion. It is possible however, that these compounds were not sufficiently concentrated to give visible reaction with these reagents.
Phenolics are well-known as antifungal, antibacterial and antiviral compounds
occur naturally in plants (Basha et al., 2006;
Leiss et al., 2009; Osman
et al., 2011). According to Ali et al.
(2009) the first step in defense mechanism in plants involves a rapid accumulation
of phenols at the infection site, which restricts or slows the growth of the
pathogen. The role of phenolic compounds in the host/pathogen interaction is
well established (Sarma et al., 2002) and constitutive
phenolics are known to confer resistance indirectly through activation of post-infection
responses in the host (Harborne, 1988). Several studies
have shown that some phenolics are inhibitors associated with non-host resistance
(Nicholson and Hammerschmidt, 1992) whereas, others
are formed or increased in response to pathogen infection and are considered
to be an important component in the defence response of the pathogen (Harborne
and Turner, 1984; Punja et al., 1985; Nicholson
and Hammerschmidt, 1992).
The role of phenolic compounds in the host-pathogen interaction is well established and constitutive phenolics are known to confer resistance indirectly through activation of post-infection responses in the host. Several studies have shown that some phenolics are inhibitors associated with non-host resistance whereas, others are formed or increased in response to the pathogen infection and are considered to be an important component in the defense response on the host to the pathogen.
The authors are very much thankful to the Sri Padmavati Mahila Visvavidyalam, Tirupati for providing the facilities.
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