Plant Protect. Sci. Vol. 54, 2018, No. 2: 00

Transcription

1 Plant Protet. Si. Vol. 54, 218, No. 2: Effet of Nutrition on Theoroma aao L. Suseptiility to Phytophthora megakarya Infetion Emile Minyaka 1,3*, Collette Vanessa Madina Banen 1, Barara Kusznierewiz 4, Oumar Doungous 2,3, Soungouna Haouni 1, Joseph Hawadak 1, Niolas Niemenak 3 and Denis Ndoumou Omokolo 3 1 Biohemistry Laoratory, Faulty of Siene, University of Douala, Douala, Cameroon; 2 Biotehnology Laoratory, Institute of Agriultural Researh for Development, IRAD Ekona Regional Researh Centre, Buea, Cameroon; 3 Laoratory of Plant Physiology and Biohemistry, Department of Biologial Siene, Higher Teahers Training College of Yaoundé, Yaoundé, Cameroon; 4 Department of Food Chemistry, Tehnology and Biotehnology, Faulty of Chemistry, Gdańsk University of Tehnology, Gdańsk, Poland *Corresponding author: minyakae@yahoo.fr Astrat Minyaka E., Madina Banen C.V., Kusznierewiz B., Doungous O., Haouni S., Joseph Hawadak J., Niemenak N., Omokolo D.N.: Effet of nutrition on Theoroma aao L. suseptiility to Phytophthora megakarya infetion. Plant Protet. Si. A new strategy to redue the severity of lak pod disease (BPD) in T. aao plants using nutrition was investigated. The dynamis of the tolerane to BPD of 18 suseptile T. aao plantlets oming from the ross ( SNK64 UPA14) was monitored during weekly (8 weeks) supply of into the soil. Prior to appliation, disease sores of the 18 plantlets (in six sets of three plantlets per set) were varying etween 3.5 (suseptile) and 5 (highly suseptile). After appliation, a sustantial derease in disease sores was oserved ompared to the ontrol. The perentage of disease tolerane gain of plantlets versus supplied ( 2.96 g) presented a quasi-hyperoli urve with asymptoti line orresponding to 6% (day 28) and 7% (day 56). Cysteine ontent was not signifiantly different etween the six triplets efore nutrition. On days 28 and 56 of supplementation, ysteine ontent presented a pattern similar to the tolerane gain of plantlet sets. The monitoring of glutathione ontent versus supplementation (ompared to day ) showed sigmoid (day 28) and hyperoli (day 56) urves whih were assoiated with defined mathematial laws determined y MALAB software. Negative and highly signifiant orrelations were oserved etween disease sores, ysteine and glutathione ontents in leaves while positive and highly signifiant orrelations were oserved etween ysteine and glutathione ontents in leaves. These data might mean that nutrition signifiantly improved the tolerane of T. aao. The mehanism of tolerane improvement might e assoiated with the synthesis of sulphur-ontaining ompounds (ysteine and glutathione) whih might e diretly or indiretly used y T. aao against P. megakarya. Keys words: ooa; profitaility; sulphur; defene; pathogen Cooa is one of the most important ash rops in Cameroon and other produing Afrian ountries (Tharuahokengo 25). Cooa ulture suffers severe yield losses due to the lak pod disease of ooa indued y P. megakarya attaks (Nyasse et al. 27). Developing strategies for the management of this disease in Cameroon are the way out to improve ooa prodution, produtivity, and profitaility. 1

2 Vol. 54, 218, No. 2: Plant Protet. Si. Two main approahes are used to protet T. aao against diseases, and these inlude: (a) the appliation of pestiides (hemials) against the pathogen P. megakarya, ut these hemials have negative impats on the human health, environment, and sustainale agriulture; () reeding whih aims to develop genetially tolerant/resistant genotypes to lak pod disease (Nyassé et al. 27). However, in T. aao, tolerane/resistane to lak pod disease (BPD) is a multigeni harater. Parents used in reeding programs for resistane to BPD are almost always heterozygotes for the harater. Consequently, the progeny is highly heterogeneous (for this harater) with most plants suseptile to BPD (Efomagn et al. 211; Nyadanu et al. 212). In the early 199s, in Sotland, Shnug et al. (1995a) reported a dramati inrease of fungal diseases simultaneously with a derease in atmospheri sulphur (S). Duuis et al. (25) demonstrated a lear link etween the sulphur nutritional status of oilseed rape and its resistane against Leptosphaeria maulans, Botrytis inerea, and Phytophthora rassiae. Moreover, these authors investigated the antimiroial ativity of plant extrats and found a very signifiant redution of antimiroial ativity in S-defiient plants that had redued gluosinolate ontent. We hypothesise that sulphur may e involved diretly or indiretly in resistane of T. aao against P. megakarya. In the asene of an effetive method of the BPD ontrol, other strategies that ould e exploited alone or in omination with geneti improvement suh as the use of nutritive Moleule whih ould Reinfore the Natural Defene System (MRNDS) are highly soliited. is a non-pollutant salt whih an e used as a soure of sulphur. It is naturally present in soil as Mg 2+, SO 4 2. is a omponent of DKW (Driver & Kuniyuki 1984) salt medium mostly used (with good results) in T. aao somati emryogenesis and plant regeneration (Minyaka et al. 28, 21). The involvement of sulphur in resistane against fungal diseases in ertain plants oupled with its importane in T. aao miropropagation emphasised the need to fous on a sulphate salt ( ) nutrition in T. aao protetion against the destrutive fungal pathogen, P. megakarya. Moreover, the suess of MRNDS would protet T. aao against more than one disease. This paper aims to study the effet of sulphate ( ) nutrition on the resistane of T. aao plants against BPD aused y P. megakarya. Material and methods Plant material. T. aao seeds from pods of SNK64 UPA14 otained y manual pollination were used to estalish a nursery. Leaves from three to four months old plantlets were used as plant iologial material in a leaf dis test to evaluate the suseptiility of T. aao plants to BPD aording to the adapted method of Nyasse et al. (1995). The most sensitive hyrids (leaf dis test disease sores etween 3.5 and 5) to BPD were used for susequent experiments. Pathogen material. The pathogen material used in this investigation was P. megakarya strain ELEG-8 (haraterised y RADP at CIRAD, Montpellier- Frane). This strain was graiously offered to us y the laoratory of plant pathology of IRAD (Institute of Agriultural Researh for Development) at Nkolisson (Yaoundé, Cameroon). In our laoratory, the strain of P. megakarya was preserved y frequent suultures on 1.5% (w/v) pea-ased agar medium. To maintain its virulene, the strain was periodially inoulated onto ooa pods. Experimental design. Eighteen of the most sensitive (leaf dis test disease sores etween 3.5 and 5) plantlets from SNK64 UPA14 were randomly seleted and plaed (in the nursery; 14 h light/1 h darkness at 26 ± 1 C) in six sets of three plantlets per set. Three plantlets of eah set reeived the same and fixed quantity of in 25 ml distilled water (every 7 days for 56 days) exept the ontrol (three plantlets) whih reeived the equivalent quantity of distilled water. The quantity of applied in a given set (of three plantlets) was defined ased on the quantity of (.74 g/l) found in DKW salt Tale 1. Different sets (6) of plantlets and quantities of supplied weekly for eah plantlet of a given set Plantlet sets S I (ontrol) S II (negative ontrol) S III (positive ontrol) S IV (test 1) S V (test 2) S VI (test 3) (g) Distilled water (ml)

3 Plant Protet. Si. Vol. 54, 218, No. 2: medium. quantities for eah set of plantlets are presented in Tale 1. The quantities were determined through the following mathematial law: Q n = Q 2 n 1 Q =.74 g; n 4 where: Q positive ontrol, quantity in g of, i.e. the quantity of in 1 l of DKW salt omplex (Driver & Kuniyuki 1985) Prior to nutrition on day, the eighteen plants (in six sets of three plants) aged 3 4 months (with 6 7 leaves) were sumitted to leaf dis tests in three independent experiments (or tripliate). Zoospore prodution. Zoospores (or inoulums) were otained aording to the Nyasse et al. (1995) adapted method. Zoospores were otained from 1-days-old ultures. Cultures with sporangia were indued to lierate zoospores y adding sterile distilled water at 4 C. After 1 h at room temperature, the zoospore onentration was adjusted to 3 l 5 zoospores/ml with Malassez hemoytometer (Assistant; Inter-Equipement, Bordeaux-Mérigna, Frane). Sreening for suseptiility of SNK64 UPA143 progeny to P. megakarya. The sreening for suseptiility of SNK64 UPA143 progeny to P. megakarya was onduted on days, 28, and 56 of sulphate nutrition. A leaf dis test was used for sreening for suseptiility of the 18 hyrids from SNK64 UPA143 progeny aording to the Nyasse et al. (1995) adapted method. The experimental design onsisted of three repliates and ompletely randomised 5 loks of leaf diss (Ø = 1.5 m) per hyrid. Hene, a total of 2 diss were used per hyrid. For eah hyrid of the progeny, leaf diss were otained from the slightly lignified young leaves (2 2.5 months old). Leaf diss were plaed in trays and inuated for 24 h (at 25 ± 1 C) in darkness prior to inoulation. After 24 h, leaf diss were inoulated y depositing 1 µl (3 1 5 zoospores/ml) of zoospore suspension on either side in the middle of eah leaf dis and inuated in darkness (at 25 ± 1 C). The nerosis rate (from tolerant to 5 highly sensitive) of suseptiility (through the nerosis size) of eah leaf dis (for eah hyrid) was registered on day 4, 5, 6, 7, and 8 after inoulation. Cysteine and glutathione extration. Simultaneously with the leaf dis test sreening, ysteine was extrated from leaves of the 18 hyrids on days, 28, and 56 of nutrition. Leaves 2.5 months old were slightly ground in a mortar in the presene of 5 ml aetone (to remove hlorophyll) and dried for 5 min at room temperature on Whatman Nº1 filter paper. For ysteine extration,.5 g of hlorophyll-free leaves was ground in the presene of 2.5 ml of ethanol 8º and entrifuged for 3 min at 6 g. The supernatant was olleted for ysteine quantifiation. Glutathione was extrated y grinding.5 g of hlorophyll-free leaves in 2.5 ml of Tris-HCl uffer (5 mm, ph 7.4) followed y entrifugation (3 min, 6 g at 4ºC). The supernatant was used to quantify glutathione in leaves on days, 28, and 56 of nutrition. Cysteine and glutathione quantifiation. Cysteine ontent was determined aording to the Gaitonde (1967) method. Cysteine extrat (.15 ml) was mixed with.35 ml of aidi ninhydrin reagent [1.3% (w/v) ninhydrin in 1 : 4 onentrated HCl : CH 3 COOH]. The mixture was heated at 1 C for 1 min, then it was ooled in ie ath to allow PING olour development. The optial density was read at 56 nm against the ontrol in whih.15 ml of ysteine extrat was replaed y an equal volume of ethanol 8. Glutathione was quantified using DTNB/EDTA aording to the Elman (1959) method. 5 µl of phosphate uffer (1 mm ph 6.8) ontaining 8 mm DTNB and 19 mm EDTA were mixed with 1 µl glutathione rude extrat and 1 ml of Tris-HCl uffer (.5 M ph 7.4) and inuated for 25 min at room temperature. The optial density was read at 412 nm against the ontrol in whih the rude extrat was replaed y 1 µl of Tris-HCl uffer (5 mm, ph 7.4). Data analysis. Colleted data were firstly sujeted to desriptive statistis and analysis of variane (ANOVA). Then, the means were separated using the Student-Newman-Keuls test (at 5% signifiane level). Spearman s orrelation analysis etween variales ( mass, disease sores, ysteine and glutathione ontents on days, 28, and 56) was onduted to evaluate the dependene etween these variales. These statistial analyses were performed y SPSS v17. software. Data on glutathione ontent in leaves versus quantities of supplied into the soil were onverted in a mathematial model (mathematial equation) using MATLAB software. Results The effet of weekly exogenous nutrition on T. aao tolerane to P. megakarya was monitored simultaneously with ysteine and glutathione 3

4 Vol. 54, 218, No. 2: Plant Protet. Si. Disease sores Day Day 28 Day 56 a a a a a a a a a a a a a a a a a a a P1 P2 P3 P4 P5 P6 P7 P8 P9 P1 P11 P12 P13 P14 P15 P16 P17 P18 T. aao plants Figure 1. Disease sores of T. aao plants (genotypes) on day, day 28, and day 56 of nutrition Plantlets are ranged in sets of three plantlets reeiving the same quantity of weekly; sets of plantlets reeived inreasing quantities of ; values are expressed as means and standard deviations; values that are signifiantly different for a given plantlet (in same set of three plantlers) at the 5% level of signifiane are indiated with different letters ontents in young leaves of T. aao on days, 28, and 56 of supply. Dynamis of the tolerane (to P. megakarya) of T. aao plants during exogenous sulphate supply to the soil. On day (efore sulphate supply as ), 55.6% of plants presented the highest disease sore (5) while 38.86% showed a disease sore of 4 and 5.56% indiated a disease sore of 3.5. When sulphate was supplied four times (day 28), there was a sustantial derease in plant suseptiility to P. megagarya in the five sets of plants used as the test ones while there was no hange in plants used as the ontrol. The derease in plant suseptiility was amplified when additional four sulphate doses were applied to soil (day 56) (Figure 1). The derease in disease sore versus was followed on days, 28, and 56. It appears that on day there was no signifiant differene etween disease sores (mean value otained from disease sores of three plantlets of eah set) of the six sets of plantlets. Four-week (day 28) or eight-week (day 56) supply of indued a signifiant derease in disease sore while supply inreased (Figure 2). The perentage of disease tolerane (ompared to day ) as a funtion of supply to soil was represented y a quasi-hyperoli urve for day 28 and day 56 with the respetive highest value of 54.11% and 65.88% for days 28 and 56 of supply. The asymptoti line seems to orrespond to the values 6 and 7% for days 28 and 56, respetively (Figure 3). Dynamis of ysteine ontent in leaves during exogenous supply. Cysteine ontent was analysed on days (efore ), 28 (after 4 times ), and 56 (after 8 times supply) in the six triplets of plants with gradually inreasing ontents of. On day, ysteine ontent of individual plants was variale from one plant to another. The addition of in test triplets of 6 7 Means of disease sores of plants triplets Day Day 28 Day 56 Tolérene (%) Day 28 Day Quantity of supplied weekly (g) Figure 2. Profile of the means of disease sores versus supplied into the soil weekly Disease sores are means ± SD (n = 28 3) Quantity of supplied weekly (g) Figure 3. Plant tolerane hanges (%) susequent to supplied into the soil Disease sores are means ± SD (n = 28 3) 4

5 Plant Protet. Si. Vol. 54, 218, No. 2: Cysteine (µg/g of dry weigth) Day Day 28 Day (g) Figure 4. Cysteine ontent versus supplied into the soil Cysteine ontents are means ± SD (n = 3 3) Cysteine (µg/g of dry weigth) Day 28 Day Day 56 Day (g) Figure 5. Inrease in ysteine ontent (day 28 day and day 56 day ) versus sulphur (from ) ontent Cysteine ontents are means ± SD (n = 3 3) plants for four times (day 28) showed a signifiant inrease of the sulphurous amino aid in eah plant exept the three plants used as ontrol. Four more additions (day 56) of inreased the ysteine ontent in leaves (Figure 4). Curves of the means of ysteine ontents of eah triplet of plants reeiving the same quantity of as a funtion of inreasing supply showed no signifiant differene etween triplets on day. Reversely, the fourfold supply of (day 28) resulted in an inrease in ysteine ontents as the supply to soil inreased from one triplet of plants to another. The same shape was oserved when four additional doses of were supplied (day 56). However, the shapes of oth urves (day 28 and day 56) were not linearly proportional to the mathematial law of supply. In fat, day 28 and day 56 urves appeared hyperoli while the supply mathematial law is an exponential funtion (Figure 5). Dynamis of glutathione ontent in leaves during exogenous supply. Glutathione ontent in leaves was evaluated on day of supply in 18 (in 6 triplets of) T. aao plants. It appeared that the ontent of glutathione in young leaves of the 6 triplets of plants was not signifiantly different etween triplets. Four times supply (day 28) led to a signifiant inrease in glutathione ontent (ompared to the ontrol where was not applied to the soil). On the day 56 of nutrition, glutathione ontents in leaves were more onsistent ompared to days and 28. However, the graphs of glutathione ontents in leaves as funtions of inreasing supply had almost the same pattern on days 28 and 56 (Figure 6). Glutathione (µg/g of dry weigth) Day Day 28 Day (g) Glutathione (µmol) Day 28 Day.4 Day 56 Day Sulphur (mole) Figure 6. Glutathione ontent versus supplied into the soil Cysteine ontents are means ± SD (n = 3 3) Figure 7. Inrease in gluthatione ontent (day 28 day and day 56 day ) versus sulphur (from ) ontent Cysteine ontents are means ± SD (n = 3 3) 5

6 Vol. 54, 218, No. 2: Plant Protet. Si. Dynami differenes (day 28 day and day 56 day ) in glutathione ontent and mathematial laws assoiated. Curves for the differenes in glutathione ontents etween day 28 and day on the one hand and day 56 and day on the other hand showed quasi sigmoid and hyperoli shapes (Figure 7). When data of these urves were sumitted to MATLAB software analysis the following mathematial laws were otained: F day28 day (x) = 1 x 3 1 x x for day28 day (1) F day56 day = (x) = 1 x 3 1 x x for day56 day (2) where: F day28 day (x) glutathione ontent in µg/g of leaves; x quantity of in the soil Both mathematial laws are similar and indiate that at the limit threshold of, glutathione ontents eome onstant. Additionally, glutathione ontent in leaves is not linearly proportional to supply. Correlation etween ysteine, glutathione ontents, supply, and plant tolerane to P. megakarya. Spearman s orrelation test showed negative and highly signifiant orrelations etween supplied to soil, ysteine ontents, glutathione ontents, and disease sores. While a positive and highly signifiant orrelation was oserved etween the quantity of supplied and ysteine, glutathione ontents on days 28 and 56 of supply. A positive and highly signifiant orrelation exists etween ysteine and glutathione on days 28 and 56 (Tale 2). Tale 2. Spearman s orrelations etween ysteine, glutathione ontents, supply, and plant tolerane to P. megakarya Mass Mass Correlation oeff 1. Sig. (2-tailed). ds day ds day Correlation oeff.695 ** 1. Sig. (2-tailed).1. ds day 28 ds day 28 Correlation oeff.874**.763 ** 1. Sig. (2-tailed)... ds day 56 ds day 56 Correlation oeff.937**.77 **.818 ** 1. Sig. (2-tailed)..1.. ys day ys day Correlation oeff Sig. (2-tailed) ys day2 8 ys day 28 Correlation Coeff.834**.612 **.663 **.817 **.5 1. Sig. (2-tailed) ys day 56 ys day 56 Correlation oeff.96**.668 ** **.97 ** ** 1. Sig. (2-tailed) gsh day gsh day Correlation Coeff Sig. (2-tailed) gsh day 28 gsh day 28 Correlation oeff.956**.726 **.891 **.94 ** **.918 ** Sig. (2-tailed) gsh day 56 gsh day 56 Correlation Coeff.981**.643 **.871 **.934 ** **.958 ** ** 1. Sig. (2-tailed) Mass mass of supplied; dsday: disease sore on day of supplementation; ds day 28 disease sore on day 28 of supplementation; dsday56: disease sore on day 56 of supplementation; ys day ysteine ontent on day of supplementation; ys day 28 ysteine ontent on day 28 of supplementation; ys day 56 ysteine ontent on day 56 of supplementation; gsh day glutathione ontent on day of supplementation; gsh day 28 glutathione ontent on day 28 of supplementation; gsh day 56 glutathione ontent on day 56 of supplementation; **orrelation is signifiant at the.1 level (2-tailed); *orrelation is signifiant at the.5 level (2-tailed) 6

7 Plant Protet. Si. Vol. 54, 218, No. 2: Disussion Sulphur, an essential element in plants, is provided mainly from the root asorption of sulphate (Kataoka et al. 24). After asorption, sulphate is distriuted into different plant organs, tissues, ells and organelles where it is sujeted to a redutionassimilation proess that leads to variale sulphurontaining ompounds (primary and seondary metaolites) with variale iologial funtions in plants (Saito 24). In the present study we monitor the effet of exogenous sulphate (as ) nutrition on T. aao against an oomyete, P. megakarya, the most destrutive pathogen of ooa prodution in ooaproduing ountries of entral and western Afria. Simultaneously with exogenous nutrition, disease sores, ysteine and glutathione ontents were monitored in young leaves of T. aao plants from the same progeny ( SNK64 UPA143). In plants, ontents of ysteine and glutathione are onsidered as markers of primary sulphate assimilation and stress response (Kruse et al. 27). The 18 plants (in six sets) from SNK64 UPA143 progeny tested for their suseptiility to lak pod disease prior to sulphate nutrition showed heterogeneity in disease sores etween plantlets. This heterogeneity is due to the fat that tolerane or suseptiility of T. aao to BPD is a polygeni harater and T. aao lones used as parents to generate a progeny are always heterozygotes for this harater (Nyassé et al. 23; Pokou et al. 28). When plantlets were sujeted to sulphate nutrition, their disease sores signifiantly dereased with inreasing quantities of. This might indiate that, when sulphate is supplied, T. aao plantlets gain tolerane or resistane to lak pod disease. However, the gain of tolerane/resistane (evaluated in perentage) was not linear to the quantity of supplied in the soil. Instead, the urve (of tolerane gain versus supplied) appeared to e hyperoli. However, the gain of tolerane/ resistane of plantlets seems to e assoiated with the quantity of supplied into the soil. This set of results might reveal the role of in the tolerane/resistane of T. aao against BPD (due to P. megakarya). The enefiial effet of in the protetion of T. aao against BPD is surely provided y SO 4 2 or sulphur. In fat, Williams et al. (22) reported an aumulation of sulphate in the vasular tissues and leaves of resistant ultivars of tomato. To explain this oservation, Cooper and Williams (24) rought up a hypothesis of sulphite (SO 3 2 ) oxidation in plant ells, leading to elementary sulphur whih has een reported as an indued antifungal sustane in plant defene (Cooper & Williams 24). Besides the elementary sulphur and sulphate, these authors also hypothesised the impliation of organi moleules ontaining sulphur suh as ysteine and glutathione in plant defene (Cooper & Williams 24). Hene, this positive impat of on T. aao protetion against BPD might also result from the impliation of sulphur in the defene platform operations in plants when exposed to ioti stress (Raush & Wahter 25; Ströher & Dietz 26). The monitoring of ysteine ontents (in the six sets of plantlets of our experimental design) indiated that efore sulphate nutrition, the ontent in ysteine was quasi idential in the six sets of plantlets whih did not present a signifiant differene in disease sores (etween sets on day ). The supplementation of sulphate into the soil resulted in a gradual inrease in ysteine ontents in plants leaves. This result firstly indiates that sulphate supplied into the soil was asored and assimilated in ysteine. However, the sulphate was supplied following an exponential mathematial law (equation) while ysteine ontent in plantlet (in sets of three plantlets) leaves showed a hyperoli urve whih might e assoiated with a logarithmi mathematial law (equation). These oservations should mean that, in T. aao plants, sulphate asorption and assimilation proesses to produe ysteine are regulated. This ould also mean that ysteine synthesised under availaility of sulphate is partially used for the iosynthesis of other sulphur-ontaining moleules in T. aao plants. The omparison of ysteine ontents and disease sore patterns in the six sets of plantlets showed that oth variales presented opposite patterns. The inrease in ysteine ontent in T. aao plantlet leaves was assoiated with a derease in disease sores of T. aao plantlets. This might highlight the protetive inidene of ysteine against ioti stress due to P. megakarya. T. aao plants might therefore use a ysteine pool to reinfore their tolerane to or ontrol of P. megakarya. This finding, reported here in T. aao for the first time, means that the onentration of ysteine in T. aao tissues might diretly e related to the tolerane/resistane of genotypes (hyrids) to lak pod disease. In tomato plants, Vidhyasekaran (2) reported a losed link etween the amino aid 7

8 Vol. 54, 218, No. 2: Plant Protet. Si. pool in plant tissues and the suseptiility of plants to pathogens. Resistant (to powdery mildew) tomato plant tissues were rih in sulphur-ontaining amino aids. Zook and Hammershmidt (1997) attested that in Araidopsis thaliana ysteine indues the synthesis of some phytoalexins suh as amalexin. In defined pathosystems and ontrolled nutritional onditions Kruse et al. (27) reported an ativation of plant sulphur metaolism in several inompatile and ompatile interations. Contents of ysteine and glutathione as markers of primary sulphate assimilation and stress response showed inreases in Araidopsis thaliana upon infetion, oiniding with the synthesis of sulphur-ontaining defene ompounds (Mou et al. 23). Glutathione ontent in leaves of T. aao plantlets prior to sulphate nutrition showed a pattern similar to that of ysteine ontent efore sulphur supply in soil. During the sulphate nutrition, glutathione ontent (day 28 day or day 56 day ) versus the quantity of supplied sulphate presented hyperoli (day 28 day ) or quasi-sigmoid (day 56 day ) urves whih might testify the iologial regulation of glutathione pool in T. aao plant tissues. As oserved with ysteine, glutathione ontent and disease sore presented antagonist patterns. Showing that, the pool of glutathione in T. aao plant tissues leads to an improvement of the tolerane of ooa plantlets to P. megakarya. Therefore, it ould e assumed that sulphate supplied into soil led to an inrease in the glutathione pool in T. aao plant tissues; and the high pool of glutathione in tissues partiipates in the tolerane/resistane of ooa genotypes (hyrids) to P. megakarya. This finding with T. aao reminds the redox-ative properties of glutathione (GSH/ GSSH) whih fulfils protetive funtions when plants are exposed to ioti or aioti stress (Raush et al. 27). Moreover, Bloem et al. (24, 27) reported that the infetion of Brassia napus L. with Pyrenopeziza rassiae inreased ysteine and glutathione ontents, as well as the ativity of l-ysteine desulfhydrase. It ould therefore e assumed that the tolerane of T. aao hyrids to P. megakarya depends on the availaility and moilisation aility of sulphate, ysteine, and glutathione whih are used for the synthesis of sulphurous antiioti moleules (metaolites, peptides, ) against the ioti stress due to P. megakarya. Therefore, as with B. napus, T. aao might moilise sulphur, ysteine, and glutathione to reat to P. megakarya infetion. This hypothesis is aked up through the negative and signifiant orrelations oserved etween disease sores and ysteine and glutathione ontents. Conlusion Sulphate supplied (as ) into soil improved the tolerane of T. aao genotypes to P. megakarya. The improvement of the tolerane is assoiated with the inrease of ysteine and glutathione pool in T. aao plants. Therefore, disease (BPD) inidene due to P. megakarya on T. aao ould e minimised (up to 7%) through the sulphate nutrition whih reinfores the defene system of this eonomially important plant. For the T. aao defene against P. megakarya, sulphur appears to e used diretly or indiretly through ysteine, glutathione or others sulphur-ontaining defene ompounds suh as antimiroial small ysteine-rih peptides alled defensins and thionins. Referenes Bloem E., Riemenshneider A., Volker J., Papenrok J., Shmidt A., Sala I., Haneklaus S., Shnug E. (24): Sulphur supply and infetion with Pyrenopeziza rassiae influene l-ysteine desulphydrase ativity in Brassia napus L. Journal of Experimental Botany, 55: Bloem E., Haneklaus S., Sala I., Wikenhäuser P., Shnug E. (27): Fats and fition aout sulphur metaolism in relation to plant-pathogen interations. Plant Biology, 9: Cooper R.M., Williams J.S. (24): Elemental sulphur as an indued antifungal sustane in plant defene. Journal of Experimental Botany, 55: Driver J.A., Kuniyuki A.H. (1984): In vitro propagation of paradox walnut rootstok. Hortiulture Siene, 19: Duuis P.H., Marazzi C., Staedler E., Mauh F. (25): Sulphur defiieny auses a redution in antimiroial potential and leads to inreased disease suseptiility of oilseed rape. Journal of Phytopathology, 153: Ellman G. (1959): Tissue sulfhydryl groups. Arhive of Biohemistry and Biophysis, 82: Gaitonde M. (1967): A spetrophometri method for the diret determination of ysteine in the presene of other naturally ourring amino aids. Biohemistry Journal, 14: Kataoka T., Hayashi N., Yamaya T., Takahashi H. (24): Root to-shoot transport of sulphate in Araidopsis. Evidene for the role of SULTR3;5 as a omponent of low- 8

9 Plant Protet. Si. Vol. 54, 218, No. 2: affinity sulphate transport system in the root vasulature. Plant Physiology, 136: Minyaka E., Niemenak N., Issali E.A., Sangaré A., Omokolo N.D. (21): Sulphur depletion altered somati emryogenesis in Theoroma aao L. Biohemial differene related to sulphur metaolism etween emryogenei and non emryogeni alli. Afrian Journal of Biotehnology, 9: Minyaka E., Niemenak N., Fotso, Sangare A., Omokolo N.D. (28): Effet of and K 2 SO 4 on somati emryo differentiation in Theoroma aao L. Plant Cell Tissue Organ Culture, 94: Mou Z., Fan W., Dong X. (23): Induers of plant systemi aquired resistane regulate NPR1 funtion through redox hanges. Cell, 113: Nyassé S., Cilas C., Hérail C., Blaha G. (1995): Leaf inoulation as early sreening test for ooa (Theoroma aao L.) resistane to Phytophthora lak pod disease. Crop Protetion, 14: Nyassé S., Efomagn M.I.B., Kéé B.I., Tahi G.M., Despréaux D., Cilas C. (27): Integrated management of Phytophthora diseases on ooa (Theorama aao L.): impat of plant reeding on pod rot inidene. Crop Protetion, 26: Nyassé S., Efomagn M.I.B., Bouami E., Ndoume-Nkeng, M., Eskes A.B. (23): Early seletion for resistane to Phytophthora megakarya in loal and introdued ooa varieties in Cameroon. Tropial Siene, 43: Pokou N.D., N Goran J.A.K., Kee I., Eskes A., Tahi M., Sangare A. (28): Levels of resistane to Phytophthora pod rot in ooa aessions seleted on farm in Cote d Ivoire. Crop Protetion, 27: Raush T., Wahter A. (25): Sulphur metaolism: a versatile platform for launhing defene operations. Trends in Plant Siene, 1: Raush T., Gromes R., Liedshulte V., Müller I., Bogs J., Galovi V., Wahter A. (27): Novel insight into the regulation of GSH iosynthesis in higher plants. Plant Biology, 9: Saito K. (24): Sulphur assimilatory metaolism. The long and smelling road. Plant Physiology. 136: Shnug E., Haneklaus S., Booth E., Walker K.C. (1995): Sulphur supply and stress resistane in oilseed rape. In: Rapeseed Today and Tomorrow. Proeedings 9 th International Rapeseed Congress, July 4 7, 1995, Camridge, UK: Ströher E., Dietz K.-J. (26): Conepts and approahes towards understanding the ellular redox proteome. Plant Biology, 8: Tharuahokengo N. (25): Cooa prodution in Cameroon. In: Brooks K.N., Ffolliott P.F. (eds): Moving Agroforestry into the Mainstream. 9 th North Amerian Agroforestry Conferene Proeedings, June 12 15, 25, St. Paul, USA. Vidhyasekaran P. (2): Physiology of Disease Resistane in Plants. Vol. II. Boa Raton, CRC Press, In. Williams, J.S., Cooper, R.M. (24): The oldest fungiide and newest phytoalexin a reappraisal of the fungitoxiity of elemental sulphur. Plant Pathology, 53: Williams J.S., Hall S.A., Hawkesford M.J., Beale M.H., Cooper R.M. (22): Elemental sulphur and thiol aumulation in tomato and defense against a fungal vasular pathogen. Plant Physiology, 128: Zook M., Hammershmidt R. (1997): Origin of the thiazole ring of amalexin, a phytoalexin from Araidopsis thaliana. Plant Physiology, 113: Reeived: Aepted after orretions: Pulished online: