on the improved vigour of the root-knot nematode infected
TRANSCRIPT
Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 97, No. I, January 1988, pp. 103-109.:0 Printed in India.
On the improved vigour of the root-knot nematode infected cowpeaplant with phenols of palmyrah wood sawdust extract, fortified withgibberellic acid and indoleacetic acid
S KANNAN and A BALAJI*Department of Zoology, Thiagarajar College, Madurai 625009, India·Present Address: Department of Zoology, Madura College, Madurai 625011, India
MS received 8 April 1987; revised 14 July 1987
Abstract. With NPK solution as the basic nutrient applied to the root-knot nematodeinfected cowpea plants, amendments were made with 100 ppm of phenol prepared fromalcoholic extract of palmyrah wood sawdust. Further amendments consisted in addinggibberellic acid or indole acetic acid or gibberellic acid + indoleacetic acid. Amendednutrient applications reduced galling, fecundity of the pathogen, pathogenic impact,improved root weights and synthesis of metabolites which signified the improved vigour ofthe infected host.
Keywords. Meloidogyne incognita; Vigna unguiculata; sawdust extract; gibberellic acid;indole acetic acid; vigour.
1. Introduction
While the various enzymes in plant parasitic nematodes account for the tissue lysis(Roy 1979, 1980, 1981), many of the breakdown products like phenols, uronic acid complexes and disaccharides exhibit compatibility with the host plant tissue metabolitesand also with the nematode's enzymes, resulting in altered substrate relations andcatalytic repression of the nematode's enzyme (Feldman and Hanks 1971; Giebel1974, 1982; McIntyre 1980; Wallace 1973), such that the feeding and subsequentprogeny output of nematode are lowered, with consequent lesser pathogenic impact.This is finally reflected as tolerance in the susceptible plants. These observations ledto the reckoning of phenols, proteins, etc.. as the possible mechanisms involved inresistance during nematode infections in plants (Giebel 1974, 1982). At the same timeaccumulation of (indole acetic acid, IAA) indole compounds (Balasubramanian andRangaswami 1962; Setty and Wheeler 1968) had also been suspected to be one of thepossible reasons for galling in root-knot nematode infections. The higher level ofIAA in the galled zones and the normal tissue differentiation adjacent to the gallsand the varied influences of IAA and gibberellic acid (GA) in plant pathogenesis(Goodman et al 1967) reflect the active IAA-GA relations in the pathological metabolism. While breeding for nematode resistant crops is a welcome feature, resistancebreaking biotypes of nematode do also evolve and hence the problem goes unabated.Towards containing the infection, attempts are being made in improving the vigour/tolerance of the susceptible hosts.
Singh and Sitaramaiah (1967, 1971) stated that the nemastatic factors attributableto sawdust as organic amendment were due to the phenolics released from sawdust.McIntyre (1980) suggested that the phenols can condense with proteins of thesubstrate and also with the enzymes of nematode, leading to the altered situation of
103
104 S Kannan and A Balaji
starved nematodes, and hence lesser catalysis and better performance by the plantand reflected as improved tolerance.
In our laboratory, free phenol in varying dilutions in water, inhibited the hatchingof Meloidogyne incognita eggs. As a followup, it was observed that in 4 Meloidogynesusceptible hosts, phenol at 100 ppm in 2% N PK solutions reduced the pathogenicimpact, with altered fecundity rates of the nematode, ultimately resulting inimproved performances in those hosts. Further, in one susceptible host (cowpea),whereas 1: 1000 dilution did not appreciably retard the pathogenic impact, andwhereas I : 10 retarded the plant growth, 1: 100 dilution yielding 100 ppm of phenolchecked the infection, without affecting the host plant.. In view of the lastobservation, experiments were conducted to assess the efficacy of phenols from thealcohol extract of palmyrah wood sawdust, as well as the influence of hormone andauxin, viz. GA and IAA on the root-knot nematode pathogenesis in cowpea.
2. Materials and methods
Seeds of the cowpea (Vigna unguiculata) were surface sterilized with O·I~'-;; HgClzwashed with sterile water and sown in 10 cm diameter pots containing river sandonly, to eliminate the influence of intrinsic phenol factors in the garden soil. Thirtypots were filled with sand. sterilized at 20lbs pressure for 2 h and cooled. The soilswere irrigated with I ~~ NPK (commercial) fertilizer solution. This basic nutrient waslater amended with phenol, GA or IAA or both. Each plant was inoculated at 2 leafstage with 1100 infective juveniles of root-knot nematode, M. incoqnita. Fifty mI ofthe amended nutrients were applied twice weekly with intermittent NPK wash.
2.1 Treatments
The following 6 treatments were introduced in 5 replicates
(i) CUT
(ii) lUT(iii) IT(iv) IT(v) IT z(vi) IT 3
Control (uninoculated with nematode), untreated (1% NPKsolution only, basic nutrient).Infected (inoculated with nematode), untreated.Infected, treated (100 ppm phenol per 50 ml of basic nutrient).Infected, treated + GA - 0·05 mg.Infected. treated + IAA - 0-05 mg.Infected, treated + (IAA + GA) -0'10 mg.
Seventyfive g of palmyrah wood sawdust were refluxed for 1 h with 375 ml ofabsolute alcohol, filtered and the filtrate used as source of phenol.
Plants were allowed to stand for 30 days, after which the assay of the pathogen'spopulation, reproductive rate etc and the biochemical parameters of the hostsuch as sugars (Seifter et aI1950), lipids (Bragdon 1951), proteins (Lowry et aI1951),phenols (Bray and Thorpe 1954) and redox-enzymes (Karman 1967a, b, 1968) wereinvestigated.
2.2 N ematode population
The following parameters were employed for evaluation:
fducti (R R (PJ /g root wt)/female!g root wt
(i) Rate 0 repro uction )=~'----=---,--'-----:-::--:---'--='--1 x 100
Sawdust extract-growth hormone/auxin in the control of root-knot nematode 105
(ii) Rate of population increase (R PI)PJ-Pi
Pi X post inoculation periods (30 days)
(iii) Environmental resistance factor (encountered by the nematode)(ERF)= RRjRPI.
Pr is the final nematode population which includes eggs x eggmasses + juveniles inthe plant and the soil after 30 days of infection and Pi is the initial inoculum ofnematode.". per g weight = total number of ..~ required to produce the PJ based oneggs per cggmass assuming that each eggmass is the product of one female developedfrom one juvenile. The soil population was estimated by screening 100 g aliquots ofsoil through a meshset and counting. The population in the plant was estimated byblending 109 aliquots of shredded infected roots, followed by screening and countingof the larvae and also the eggs in the egg masses.
3. Results and Discussion
Statistical analysis of critical difference at 5 and I~.~ indicated that the growth (weightof the root tissue) and biochemical parameter of the host plant (V. unquiculatai andpopulation buildup of M. incoqnita were more significantly influenced by all thetreatments.
3.1 Patlioqenic impact
In the infected plant (IUT) receiving the NPK solution only as the basic nutrient,a progressive increase in the root weight was observed when phenol, GA, JAA,GA + IAA were introduced (table I). Among the amending factors GA + JAA withphenol resulted in the highest increase. Contrary to the increase in root weight,galling was reduced with phenol introduction, the reduction occurring further andfurther as GA, IAA or GA + IAA were employed along with phenol (table 1). ThusGA and IAA separately and also together with phenol reduced root-knot index andthe galling phenomenon. The reduction in the galling can be linked with the nematodes population turnover.
The significant impact of phenol + GA + JAA (treatment IT 3) consisted in thefollowing, as evaluated against the infected plant which received only the NPK(treatment IUT). In IT 3' there was significant reduction in galls to 1/6, eggmass to1/5, eggs per eggmass to 1/7, root and soil population to 115, over that obtained inIUT. The reproducti ve rate depressed from 3·65 (JUT) to 2·47 and rate of populationincrease depressed from 0·4521 (JUT) to 0·0462 in IT 3' indicating a 1/9th value onlyin IT 3 as against IUT. The ERF increased from 8·073 in JUT to 53·579 in JT3' i.e.more than 6 times in the presence of phenol and GA + JAA.
It was therefore evident that, while phenol alone exerted a depressive effect on thenematode, introduction of GA or IAA or GA + IAA exerted further depression bydrastically reducing the nematode's fecundity, root-knot index and galling phenomena. Consequently the root weight also increased.
Since the nematode's fecundity follows feeding, the influence of the host environs
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Sawdust extract-growth hormone/auxin ill the control of root-knot nematode 107
are well reflected. A progressive increase in sugars, lipids, proteins, phenols andredox-enzyme activities was found. indicating the dynamics of pathological metabolism of the infected plants under various nutrient treatments, with the control checksas the standard for evaluation of such events (CUT and IUT, table 2).
While the sugar reduction, common in root-knot nematode infections, are reflected in the infected plant under NPK influence only (CUT and IUT), sugar levelsincreasing with phenol application to start with, gradually showing further increments with GA, IAA, GA + IAA applications (highest in IT3)' This indicate thebuildup of carbon reserves as sugars etc. under these amendments and also perhapstheir depressed consumption by the nematode in the presence of phenol which isbelieved to cause enzyme repression in the nematode's enzyme.
The biological organization shows a preferential utilisation of proteins and sugarsfor energy transduction (Fruton and Simmonds 1939; West et al 1968) so that, theseare utilized faster than fats which serve as final endogenous energy store. It is quitelikely that. the impediments caused by phenol in condensing with the plant's metabolites such as glycosides and proteins etc. reduce the readily available energy insugars for the nematodes. Enzyme repression in nematode can also be brought aboutby the phenol and this might be responsible for the retarded nematode fecundity inphenol treatment to plants. Further, the high level of protein might be anotherfactor, perhaps not wholly conducive for the nematode.
Coupled with the above are the defence attributes attached to proteins andphenols, in the plant's resistance during pathogenesis, as well as the high energy playreflected in the enhanced redox-enzyme activities (Goodman et al 1967).
A perusal of the redox-enzyme activities shows a specific increase of dehydro-
Table 2. Biochemical components and enzyme activities in V. unyukulaca under root-knotnematode infection and amendments with nutrients.
Total Total endoge-dehydrogenase nous reductase
activities activitiesSugar Protein Lipid Phenol(mg/g) (rng/g) (mg/g) (mg/g) (mg TIC reduced/g dry weight)
CUT 6-21 ±0'08 3-05±0·04 80·19±0·42 3-01±0'01 6-638 ± 0·179 21-070±0'778
Jnfected plants with various nutrient treatmentsIUT 2-58±0-03 3-50±0-06 96-56 ± 9·15 4-08 ± 0-05 7-978±0'382 22·270±0·919
(- 58-45)* (+ 14-75)* (+ 20-41)* (+ 35'55)* (+20-19)* (+ 5'70)*
IT 4-25 ± 1·25 4·89±0·41 135'11 ± 18-40 6-09±0'06 12'818±0-336 23-600 ± 0'990( +64,73) (+ 39'71) (+ 39-92) ( +49,26) (+ 60-67) (+ 5-97)
IT, 5·63 ±0-88 6·14±0·10 149-23± 3-64 7'22±0-74 17-568± 0-983 28-670='= 1·485(+ 118-22) (+ 75'43) (+ 54,55) ( +76-96) (+120'21) (+ 28'74)
IT, 7-06±0'91 7·54±0·42 162-40± 1'7! 8-88± 1·61 20·645± 1·229 33-500± 1-980(+- 173-64) (+ 115-43) (+68,19) (+ 117,65) (+ 158'77) (+50-43)
IT,) 10·20±0·15 9·05 ±0-12 188-73±2'15 9-79±0'56 23-793 ± 1-423 44·970± 3'536(+ 295,35) (+ 158,57) 1+95-45) (+139'95) (+ 198'23) (+101'93)
CO 5"" 105 0·36 1242 1·10 1-287 2·557CD r, 1·43 0-49 16-94 1-49 1·756 3-487
Each value (mean ± SO) represents an average of 5 observations.Numbers in parentheses indicate per cent over 1liT or *CUT.
108 S Kannan and A Balaji
genase activities in the infected host under the NPK and other amendments, beinghighest in the plant, treated with phenol +GA + IAA. This indicates greater energyyield through catalysis, due to expected increase in tissue damage by nematode.However, this seems to be offset by the increased velocities of endogenouf reductasesinvolved in synthesis. In fact, the synthetic velocities of enzymes are far morepronounced than the velocities of catalytic dehydrogenases, amounting to almost1'5-2 times greater than the lytic enzymes i.e. dehydrogenases. The resultant of thesetwo enzymes activities is quite evidenced in the increments of sugars, lipids, proteinsand phenols, w.hen phenol or other factors like GA, IAA alone or coupled areintroduced as treatments to plants. It is quite likely that the preponderance of energyproduced due to catalysis, is transducted towards significant synthetic activity,meant for repair and maintenance through the reductase play.
The above features amplify well the observations of Cowling and Horsfall (1980)who stated that, stress (dietary/pathogenic) in plants is countered through themetabolic continuum through which-reallocation of resources occur to combat thecrisis and in this direction, the host plant spends the minimal energy for the combat,conserving the rest for repair and growth. That this is so, can well be understoodwhen the redox-enzyme activities are computed against the pronounced synthesis ofmetabolites during infection. As a net effect, improved tolerance or vigour isreflected, especially under amended conditions, such as introduction of phenol etc.which have a metabolic role in the plant, with the enhanced levels of the variousmetabolites.
If tolerance can be interpreted as that signifying the capacity of the host to getloaded heavily with pathogen but yet survive, the specific reduction of pathogen'spopulations with improved weights in the host, signify improved vigour simulatingresistance relations as a result of development of post infection resistance, reflectingfunctional resistance. The reduced galling phenomenon, coupled with the improvedweight of the roots and the enhanced metabolism observed in the present studiesseem to indicate improved vigour of the host under the influence of phenol + hormone +auxin. Nemastatic factors like phenols in the sawdust extract preparations,produced out of high dilutions and coupled with minute levels of hormone andauxin, thus, can be advantageously exploited for improving the vigour of viable hostsfor better produce. The high dilution factor with low levels of hormone and auxin,also help reduce the cost of application, quite favourable for agricultural economy.
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Sawdust extract-growth hormone/auxin in the control of root-knot nematode 109
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