Messa, Cameroon. Received 19 October, 2016; Accepted 22 January, 2017

Transcription

1 Vol. 11(8), pp , August 2017 DOI: /AJPS Artile Numer: 03673C ISSN Copyright 2017 Author(s) retin the opyright of this rtile Afrin Journl of Plnt Siene Full Length Reserh Pper Effets of hitosn n snil shell power on oo (Theorom o L.) growth n resistne ginst lk po isese use y Phytophthor megkry Pul Mrtil Téné Tyo 1,2, Céile Annie Ewne 1,2, Pierre Onomo Eff 1,2 n Thée Boujeko 1,2 * 1 Deprtment of Biohemistry, University of Youné 1, PO Box 812 Ngo Ekelle, Cmeroon. 2 Lortory of Phytoprotetion n Vloriztion of Plnt Resoures, Biotehnology Center-Nkolisson, P. O. Box 3851 Mess, Cmeroon. Reeive 19 Otoer, 2016; Aepte 22 Jnury, 2017 Chitosn n hitin soures hve emerge s promising groups of iologil sustnes tht n inue plnt growth n resistne to iseses. This stuy is ime t investigting the potentil effet of hitosn n snil shell to promote oo growth n inue resistne ginst Phytophthor megkry. The results showe tht tretment of oo sees with hitosn t 1.5 to 2.0% n snil shell t 2.0% inreses the perentge of germintion n lso reues the time of germintion. 12 weeks fter plnting, series of morphologil hnges ws oserve on the plnts trete with hitosn n snil shell. Plnt height (34 to 39.5 m), lef numer (11 to 13 leves/plnt), lef re (88 to 130 m 2 ) n fresh weight of roots n shoots (25 to 44 g/plnt) inrese signifintly in the hitosn n snil shell trete soils. Pos inoulte with hitosn n/or snil shell trete soil suspensions presente very slight neroti lesions. This oul e linke to erese in the P. megkry lo of the soil suspension. Lef inoultion showe vrition mong the tretments with the lowest inex (highest level of resistne) reore in plnts trete either with hitosn or snil shell. The tretment of plnts with hitosn n snil shell power efore n fter inoultion showe higher level of phenoli ompouns thn in the ontrol plnts. Furthermore, the inoultion inue signifint umultion of proteins in the oo plnts trete with hitosn n snil shell. The level of proteins umultion fter inoultion ws higher in plnts trete with snil shell thn those trete with hitosn. In onlusion, the effet of snil shell on oo growth n resistne showe tht it is possile for snil shell power to e iofungiie n iofertilizer use in the ontrol of o Blk Po Disese in nurseries. Key wors: Theorom o, Phytophthor megkry, lk po isese, hitosn, snil shell, ioontrol gents. INTRODUCTION In Cmeroon n other Afrin ountries, oo (Theorom o) is one of the most importnt eonomil sh rops. However, its ultivtion is fe with numerous prolems suh s prsiti ttk n the insuffiieny of selete genotypes. Among the prsiti onstrints, Blk Po Disese (BPD) use y severl

2 332 Afr. J. Plnt Si. Phytophthor speies hs een the worst thret of o in West n Centrl Afri (Opoku et l., 2000). Reports hve shown tht in Cmeroon, the BPD use y Phytophthor megkry is the highest thret; n losses of up to 50-80% of oo ens hve een reporte (Noume-Nkeng et l., 2004). This isese reues the yiel n qulity of the prout, n lso inreses the ost of proution. The oo fruits, stems or roots n e infete y hlmyospores or sporngi, whih my germinte to proue swimming zoospores tht n e spre y rops of rin, win-lown rin, soil n soil wter. In orer to prevent these losses ue to P. megkry infetion, ifferent strtegies hve een evelope. Chemil ontrol using metlxyl n opperse fungiies hs een reporte to e the most effetive strtegy to reue the impt of BPD in nurseries n in frms (Noume-Nkeng et l., 2004; Sonw et l., 2008). However, pplition of fungiies n hve rsti effets on the onsumer n the environment (Nsey et l., 2000). A promising n sfer metho for ontrolling oo BPD hs een the evelopment of resistnt ultivrs n the use of pproprite ulturl prties (Nysse et l., 2007; Thmeni et l., 2011). However, isese resistnt ultivrs re not yet ville. A ioontrol pproh using hitosn or hitin soures (snil shell, r skeleton, shrimp skeleton) is n eofrienly lterntive. Chitosn is rohyrte iopolymer erive from eetyltion of hitin, whih is foun in the shells of Crustens, utiules of insets n ell wlls of fungi. The positive hrge of hitosn onfers to it numerous n unique physiologil n iologil properties with gret potentil in wie rnge of griulturl prties (Butist-Bnos et l., 2004; Tng et l., 2010). Mny stuies hve reporte the pity of hitosn to stimulte the immune system for plnt resistne to pthogen infetion, to inue the umultion of phytolexins resulting in ntifungl responses in orer to enhne protetion ginst further infetion (Coqueiro et l., 2011) n lso to hnge the soil miroorgnisms ontent (Roy et l., 2010). Moreover, hitosn hs een wiely use s growth stimultor, germintion elertor n yiel enhner in mny rop speies suh s in orhi (Uthirtnkij et l., 2007), f en (El-Swy et l., 2010), uumer (Sheht et l., 2012) n orn (Boonlertnirun et l., 2011; Lizárrg- Pulín et l., 2011). This stuy is therefore ime t investigting the ility of hitosn n snil shell power to promote oo growth n inue resistne ginst P. megkry in nurseries y evlution of the rte of germintion of sees, plnt gro-morphologil hrteristis, suppressive potentil, totl phenoli ompouns n peroxise tivities. Our finings will ontriute to the evlution of the role of hitosn n snil shell power s iofungiie lterntive in the ontrol of oo BPD. MATERIALS AND METHODS Soil The soil use in this experiment ws ollete from Youné (Centre region, Cmeroon) n re often use y frmers to prepre young oo seelings. The soil ws ir-rie n psse through 4 mm sieve efore mixing (3:1; v/v) with river sn. Chemil nlysis (orgni mtter, nitrogen, lium, mgnesium, phosphorus ontents n ph) of ry smples ws rrie out efore the ultivtion perio. The ontents of ville nutrients in the soil were: orgni mtter, 3.40%; nitrogen, 1.23%; lium, meq.g -1 of soil; mgnesium, meq.g -1 of soil; phosphorus, 3.54 mg.g -1 of soil; n ph ws 6.1. The soil-sn mix ws then utolve three times t 121 C for 30 min efore eing trnsferre into pots. Fungl strins n snil shell power proution Zoospore suspensions of P. megkry isolte EL from the ore olletion of IRAD (Institut e Reherhe Agriole pour le Développement, Youne, Cmeroon) were otine oring to Tonje et l. (2006). Sterilize n unsterilize soils were then inoulte with 10 6 zoospore.kg -1 of soil. Snil shell ws otine following the metho of Jieowno et l. (2007). The shells (from Bue, in the South west region of Cmeroon) were thoroughly wshe using tp wter n ir-rie for two ys. They were then rie in n oven n t 105 C, then poune in mortr using pestle to form the snil shell power. Sterilize n unsterilize soils were inoulte with zoospores of P. megkry t 10 6 zoospore.kg -1 of soil for 48 h efore, eing trete with 1% hitosn n snil shell power. Chitosn ws kinly provie y Professor Crole Beulieu, Centre SEVE University of Sherrooke (Quee, Cn). Evlution of germintion rte For evlution of germintion rte n plnt gro-morphologil hrters, mture oo pos ( MA12 PA150) hyris proue y mnul pollintion were ollete from the SODECAO (Soiété e Développement u Co) gene nks of Mengng Sttion (South Region, Cmeroon; Ltitue 2 90 N, Longitue E). Coo sees were extrte from the pos, wshe with istille wter n ote s shown in Tle 1. For the germintion test, 8 groups of 30 sees eh were sown per germintion try. A 2% hitosn stok solution ws prepre s esrie y Lizárrg-Pulín et l. (2011), n the 1.5% solution ws otine y pproprite ilution of the stok solution. Experimentl sees were sown t epth of 5 m in sterilize soils n the trys inute t 25 ± 1 C. Distille wter ws sprinkle on the trys every two ys to voi ryness. Germintion ws onfirme y shooting of the riule n the germintion rte evlute s in Zeng et l. (2012). *Corresponing uthor. E-mil: oujeko@yhoo.om. Tel: Author(s) gree tht this rtile remins permnently open ess uner the terms of the Cretive Commons Attriution Liense 4.0 Interntionl Liense

3 Tyo et l. 333 Tle 1. Experimentl esign of sees oting. Groups 1st 2n 3r 4th 5th 6th 7th Numer of sees Stte Untrete Trete Trete Trete Trete Trete Trete Chitosn (v/w) Control 2% 1.5% Snil shell (w/w) Control - - 2% 10% 20% 30% Soke for 12 h 12 h - - Oven-rie t 29 C for 1 h 1 h Evlution of gro-morphologil hrters The gro-morphologil hrters tht were ssesse inlue the weight of the plnt root n shoot weight, height, lef numer, length, with n re of the lef. These prmeters were ssesse every 4 weeks for perio of 12 weeks. To proue the plnts, single pregerminte oo seeling ws trnsplnte into eh plsti pot, whih ontine trete n untrete soil. Eh tretment ws in uplites of ten pots. All the pots were kept in the greenhouse n wtere with istille wter every two ys for perio of 12 weeks. During ssessment t the 4 weeks intervl, roots of hrveste plnts were wshe to remove soil prtiles n plnt height mesure with liper tip. Length n with of leves mesure with grute ruler n the weight of shoots n roots of freshly hrveste plnts then mesure seprtely. The experiment ws ompletely rnomize esign with two tretments hitosn n/or snils shells t 1% w/w, n the ontrol (tretments without hitosn n/or snil shells). The tretments without hitosn n/or snil shells re lso esigne: sterilize Soil (ss), sterilize Soil + P. megkry (ss+pm), n none sterilize Soil (nss). The tretments with hitosn n/or snils shells re esigne s: sterilize Soil (ss) + Chitosn (ss+ch), sterilize Soil + P. megkry + Chitosn (ss+pm+ch), none sterilize Soil (nss) + Chitosn (nss+ch), sterilize Soil + Snil Shell (ss+ss), sterilize Soil + P. megkry + Snil Shell (ss+pm+ss), n none sterilize Soil + Snil Shell (nss+ss). Eh tretment onsisting of three replites were repete twie. Inue resistne ssessment Young oo leves from two-month-ol plnts were ollete from the nursery in the greenhouse wshe thoroughly with tp wter n sterilize with 70% ethnol for 30 s. Sixteen lef iss of 1.5 m imeter from eh tretment were me with ork orer n replite three times. These lef iss were ple with their xil surfe upwrs in sie trys. On the other hn, six whole leves of oo were set prt in single try n rrnge oring to the protool esrie y Djogoue et l. (2007). These iss n whole leves were ll simultneously inoulte with 10 µl of 10 6 zoospore ml -1 suspension of P. megkry. In eh tretment, one ontrol me up of six whole leves n lef iss inoulte with 10 µl of sterilize istille wter in seprte try ws inlue. Inoultion ws performe on the unersie of eh lef, n the trys were inute in rk room t 25 ±1 C. Disese expression ws rte six ys fter, using the rting sle evelope y Nysse et l. (1995). This experiment ws repete twie, n the severity of isese ws etermine for eh tretment y lulting the rtio of the sum of iniviul sores to the totl numer of iss leves use. The isese severity inex use to express the resistne level (Pulin et l., 2008) ws s follows: Highly Resistnt (HR: 0 < inex 1); Resistnt (R: 1 < inex 2); Moertely Resistnt (MR: 2 < inex 2.5); Suseptile (S: 2.5 < inex 3.5); n Highly Suseptile (HS: 3.5 < inex 5). Biohemil nlyses Biohemil nlyses were rrie out following the ssessment of infetion on the whole leves. The smples involve were ut t 1 m eyon the nerosis point or eyon the mrke sr (setions with no symptoms). Smples from the sme tretments were omine. The prts of the leves from sterilize Soil (ss) tretment were omine. For iohemil nlyses, eh tretment ws repete twie. Determintion of the ontent of totl phenoli ompouns The extrtion n quntittive mesurement of the ontent of totl phenoli ompouns were performe s esrie y Djogoue et l. (2007) with moifition. Totl phenoli ompouns were extrte twie using 80% methnol. 1 g of fresh tissue of inoulte n helthy leves plnt ws groun seprtely in 10 ml of 80% methnol t 4 C. After 5 min of gittion, the groun mteril ws entrifuge t g for 5 min t 4 C. The superntnt ws ollete n the pellet ws re-suspene in 5 ml of 80% methnol followe y gittion for 5 min. After the seon entrifugtion t 4 C, the superntnt ws ollete n mixe with the previously ollete superntnt to onstitute the phenoli extrt. The onentrtion of phenoli ompouns ws etermine spetrophotometrilly t 725 nm oring to the metho of Mrigo (1973), using the Folin-Ciolteu regent. Totl phenoli ompoun ontents were expresse in mg equivlent of tehin per g of fresh weight. Determintion of the ontent of totl ntive protein For the etermintion of totl ntive protein ontent, extrtion ws performe s esrie y Priminho et l. (2008) with moifition. 1 g of fresh tissue of inoulte n helthy leves plnt ws groun seprtely in 10 ml of extrtion uffer (Tris-HCl 10 mm ph 7.5, Triton X-100 1%) t 4 C, stirre for 10 min n kept on ie. The smples were sonite (8 pulses of 3 s eh with 10 s intervls) with the setting t 70% output on n Ultrsoni proessor (Gex 130, 130 W), n then entrifuge t g for 25 min t 4 C. The pellet ws sumitte to seon extrtion. Both superntnts were mixe with 0.4 volume of n-utnol n 1/10 of 3 M NA ph 4.5. The smples were kept on ie for 30 min with gittion every 10 min, n then entrifuge t g for 15 min t 4 C. The superntnt ontining totl proteins ws store t 4 C. The proteins were quntifie using the Brfor (1976) metho. 1 ml of Brfor regent ws e to eh ml of extrt. The sorne ws mesure t 595 nm using UV-VIS 1605 Shimzu

4 334 Afr. J. Plnt Si. Figure 1. Germintion perentge of see ote n unote with hitosn (Ch, 1.5 n 2%) n snil shell (SS) t 2, 10, 20 n 30%. Eh tretment onsisting of three replites were repete twie. spetrophotometer. BSA ws use s the stnr. Peroxises tivity Peroxise tivity ws etermine y spetrophotometry t 470 nm in the totl ntive protein extrts oring to the metho of Boujeko et l. (2005) using Tris-mlete uffer n oxygente wter (H 2O 2). The enzyme tivity ws expresse in enzyme unit per g of fresh weight. Evlution of the suppressive effet of snil shell For evlution of the suppressive effet of snil shell n hitosn in the soil, nmely, P. megkry, 3 month-ol helthy pos (SNK10, suseptile lone) were hrveste, wshe with tp wter, sterilize with 70% ethnol (for 1 min), 10% (v/v) ommeril soium hypohlorite (for 5 min) n rinse 3 times with sterilize istille wter. The inoultion ws rrie out y the eposition of 500 µl suspensions of untrete n trete soils ollete fter 12 weeks of experiment on the sr otine with hn utensils. The srs re then lose with otton tht hs een immerse in sterilize wter. The soil suspension ws otine y mixing soil with sterilize istille wter. Tht is, 2 g of soil smple ws mixe with 10 ml of sterilize istille wter, shken n llowe to stn for 10 min. A ontrol onstitute of pos inoulte with only sterilize istille wter ws relize. The entire inoulte pos were inute in rk room t 25 ± 1 C in humi hmer. The rte of nerosis ws qulittively evlute on ily sis for 6 ys using the sign + or -. ifferenes were foun, pirs of smples were ompre y Tukey s test t p RESULTS Effet of hitosn n snil shell on see germintion The tretment of oo sees with hitosn t 1.5 to 20% n snil shell t 2% inreses the perentge of germintion n lso reues the time of germintion ompre to the ontrol sees (Figure 1). Tretment of sees with hitosn t 2% inrese the germintion perentge fter sowing from 0 to 40% within 7 ys (D7) n to 100% within 14 ys (D14). Tretment of sees with snil shell (SS) oting t 2% lso inrese the germintion perentge from 0 to 40% within 7 ys (D7) ut rehes 100% germintion within 21 ys (D21). The tretment with snil shell t 10 n 20% i not inrese the germintion perentge t the sme rte. Hene, with these onentrtions, the germintion perentge inrese from 0 to 60% within 14 ys (D14) n rehes 75% fter 21 ys (D21). The effet of tretment with snil shell t 30% onentrtion ws t the sme level with the ontrol sees, tht is, 40% of germintion within 21 ys (D21). Sttistil nlyses Dt nlysis ws performe using the SPSS softwre version All the results were expresse s mens ± stnr evition n sujete to Anlysis of Vrine (ANOVA). Where signifint Effet of hitosn n snil shell on gromorphologil hrteristis The gro-morphologil hrteristis of oo

5 Tyo et l. 335 Tle 2. Effet of hitosn n snil shell soil tretments on gro-morphologil hrteristis of oo seeling fter twelve weeks of growing. Tretment Numer of leves Lef re (m 2 ) Plnt height (m) Shoots (g) Roots (g) nss 7.33 ± ± ± ± ± 0.25 ss 7.33 ± ± ± ± ± 0.05 nss+ch 11.5 ± ± ± ± 0.05 g 9.55 ± 0.05 nss+ss 12 ± ± ± ± ± 0.05 e ss+ch ± ± ± ± 0.05 f ± f ss+ss 12.5 ± ± ± ± 0.88 e ± 0.01 f ss+pm 7 ± ± ± ± ± 0.05 ss+pm+ch 10.5 ± ± ± ± 0.5 e 8.82 ± 0.05 ss+pm+ss 12.5 ± ± ± ± ± 0.05 Eh tretment onsisting of ten replites ws repete twie. Mens with the sme letter within olumn re not signifintly ifferent t P nss: none sterilize Soil; ss: sterilize Soil; nss+ch: none sterilize Soil + Chitosn; nss+ss: none sterilize Soil + Snil Shell; ss+ch: sterilize soil + Chitosn; ss+ss: sterilize soil + Snil Shell; ss+pm: sterilize Soil + P. megkry; ss+pm+ch: sterilize Soil + P. megkry + Chitosn; ss+pm+ss: sterilize Soil + P. megkry + Snil Shell. seelings (height of the plnt, numer of leves, re of lef, weight of shoots n roots) grown in soils trete with hitosn n snil shell were vrily ffete in ifferent stges fter 12 weeks of growth (Tle 2). The heights of oo seelings grown in soil trete with hitosn n snil shell power were oth signifintly ifferent from those of the ontrol plnts. The ontrol plnts (nss, ss n ss+pm) elonge to the sme group with n verge height of 26 m. The plnts grown in sterilize soil (ss) n unsterilize soil (nss), trete either with hitosn (ss+ch n nss+ch) or with snil shell (ss+ss n nss+ss) h n verge height of 38 m n elonge to the sme group. The presene of P. megkry in the sterilize soil (ss) slowe own this growth in height s shown on Tle 2, with n verge height of 34 m for the tretments ss+pm+ch n ss+pm+ss. Furthermore, the numer of leves n the re of leves of oo plnts grown in the soils (nss, ss n ss+pm) trete either with hitosn or snil shell power vrie oring to the tretment fter 12 weeks of growth (Tle 2). The verge numer of leves in the ontrol plnts (nss, ss n ss+pm) ws 7 leves n ws signifintly ifferent from those of the trete plnts whih h n verge of 12 leves, with no signifint ifferene etween the tretments. The re of leves showe 2 ifferent groups: the ontrol plnts n trete plnts. The tretments without hitosn n/or snil shells (ss, ss+pm, nss) showe level of lef res signifintly lower thn those the tretments with hitosn n/or snils shells (ss+ch, ss+pm+ch, nss+ch,ss+ss, ss+pm+ss, nss+ss). The weights of fresh shoots n roots of the oo plnts grown in soils trete either with hitosn or snil shell power were signifintly ifferent etween the trete plnts n the ontrol (Tle 2). The weight of the shoots h 3 ifferent groups: the first group (nss, ss n ss+pm) h men of 14 g, the seon group (ss+ch, ss+pm+ch, nss+ch,ss+ss, ss+pm+ss, nss+ss) h men of 33 g n the thir group (ss+ch n nss+ch) h the highest. The weight of the roots h 2 ifferent groups: the first group (nss, ss n ss+pm) h men of 4 g, the seon group (ss+ch, ss+pm+ch, nss+ch,ss+ss, ss+pm+ss,nss+ss) h men of 10 g. Chitosn tretments onsistently improve the weight of the oo shoots n roots more thn tht of snil shell tretment. Evlution of isese severity The iss of oo leves inoulte with 10 µl of 10 6 zoospore.ml -1 suspension of P. megkry evelope ler lesion six ys fter, while no symptom ws seen on iss of leves inoulte with sterilize istille wter. The isese severity ws signifintly (p < 0.05) ifferent mong the tretments. The highest level of isese severity ws oserve with the ontrol tretments (ss, nss n ss+pm). While, the lowest level of isese severity ws reore in plnts trete with either hitosn or snil shell, showing isese severity inex of 0.83 to 1.15 for hitosn tretments n 1 to 1.4 for snil shell tretments (Figure 2). Biohemil nlyses The mount of totl phenoli ompouns in noninoulte plnts ws lower thn the inoulte ones. The inoultions of leves h signifint effet on totl phenoli ontents in ll the tretments. The tretment of plnts with hitosn n snil shell power efore n fter inoultion showe higher level of phenoli ompouns thn in the ontrol plnts. Chitosn tretment h more signifint effet s ompre to snil shell

6 Disese severity inex 336 Afr. J. Plnt Si e e 0 Tretments Figure 2. Disese severity of plnts trete n untrete with hitosn n snil shell six ys fter inoultion. Eh tretment onsisting in three replites were repete twie. Mens with the sme letters re not signifintly ifferent t P< ss: sterilize Soil; nss: none sterilize Soil; ss + Pm: sterilize Soil + P. megkry; ss + Ch: sterilize Soil + Chitosn; nss + Ch: none sterilize Soil + Chitosn; ss + Pm + Ch: sterilize Soil + P. megkry + Chitosn; ss + SS: sterilize Soil + Snil Shell; ss + SS: sterilize Soil+ Snil Shell; nss + SS: none sterilize Soil + Snil Shell. tretment (Figure 3). The presene of P. megkry in the soil seeme to slow own the inrese of the mount of phenoli ompouns exept for the hitosn tretment. Chitosn tretment showe n inrese of the mount of phenoli ompouns fter inoultion, with n inrese of 14, 23 n 44% for the tretments nss+ch, ss+ch n ss+pm+ch, respetively. This ws not the se with the snil shell tretment whih showe n inrese level of phenoli ompouns fter inoultion tht ws signifintly lower, regrless of the nture of tretment. The mount of proteins ws muh lower in the plnts grown in ontrol soils (nss, ss n ss+pm) efore n fter inoultion. The tretment with snil shell n hitosn inrese the protein level in helthy n inoulte plnts (Figure 4). The presene of P. megkry in the soil h no signifint effet on the protein in the plnts ompre to the other tretments. The inoultion inue signifint umultion of proteins in the oo plnts trete with hitosn n snil shell. The proteins umultion fter inoultion ws higher in plnts trete with snil shell thn those trete with hitosn (Figure 4). The peroxise tivity of plnt leves grown in ontrol soils (nss, ss n ss+pm) efore n fter inoultion ws not signifintly ifferent from tht of the tretments nss+ss efore n fter inoultion (Figure 5). In plnts trete with hitosn, the peroxise umultion ws higher in the non-inoulte plnts with n verge of 0.92 UE/min/g fresh weight, n this mount signifintly rose to 76% in ll the plnt leves fter inoultion (ss+ch, ss+pm+ch n nss+ch). Furthermore, there ws signifint ifferene in peroxise umultion etween the leves of plnts trete with hitosn n those trete with snil shell. Effet of P. megkry soil inoulum lo on oo pos The oo pos inoulte with sterile soil ontining P. megkry n non-sterile soil trete with hitosn n snil shell, respetively showe very low levels of neroti lesions tht ppere only 6 ys (D6) fter inoultion. For the tretments without hitosn n snil shell (nss n ss+pm), neroti lesions ppere n spre on the inoulte oo pos from D4 n D2 fter inoultion till D6. The pos inoulte with sterilize soil n with zoospores of P. megkry (ss+pm) got ompletely rotten (Tle 3). DISCUSSION The im of this stuy ws to ssess the ility of hitosn n snil shell power to inue growth n resistne of oo ginst P. megkry, the usl gent of BPD in

7 Proteins (mg equivlent of BSA/.g of F W) Phenolis (mg Equivlent of tehin/g F W) Tyo et l Helthy Inoulte 1 e Tretments Figure 3. Vrition of totl phenoli ontent in trete n untrete plnt efore n six ys fter inoultion. Eh tretment onsisting of three replites were repete twie. Mens with the sme letters re not signifintly ifferent t P< ss: sterilize Soil; nss: none sterilize Soil; ss + Pm: sterilize Soil + P. megkry; ss + Ch: sterilize Soil + Chitosn; nss + Ch: none sterilize Soil + Chitosn; ss + Pm + Ch: sterilize Soil + P. megkry + Chitosn; ss + SS: sterilize Soil + Snil Shell; ss + SS: sterilize Soil+ Snil Shell; nss + SS: none sterilize Soil + Snil Shell. 8 Helthy Inoulte h h g f g g f e f e f 0 Tretments Figure 4. Vrition of totl proteins ontent in trete n untrete plnt efore n six ys fter inoultion. Eh tretment onsisting of three replites were repete twie. Mens with the sme letters re not signifintly ifferent t P< ss: sterilize Soil; nss: none sterilize Soil; ss + Pm: sterilize Soil + P. megkry; ss + Ch: sterilize Soil + Chitosn; nss + Ch: none sterilize Soil + Chitosn; ss + Pm + Ch: sterilize Soil + P. megkry + Chitosn; ss + SS: sterilize Soil + Snil Shell; ss + SS: sterilize Soil+ Snil Shell; nss + SS: none sterilize Soil + Snil Shell.

8 Peroxises tivity ( UE/min/g FW) 338 Afr. J. Plnt Si Helthy Inoulte Tretments Figure 5. Vrition of totl peroxises tivities in trete n untrete plnt efore n six ys fter inoultion. Eh tretment onsisting of three replites were repete twie. Mens with the sme letters re not signifintly ifferent t P< ss: sterilize Soil; nss: none sterilize Soil; ss + Pm: sterilize Soil + P. megkry; ss + Ch: sterilize Soil + Chitosn; nss + Ch: none sterilize Soil + Chitosn; ss + Pm + Ch: sterilize Soil + P. megkry + Chitosn; ss + SS: sterilize Soil + Snil Shell; ss + SS: sterilize Soil+ Snil Shell; nss + SS: none sterilize Soil + Snil Shell. Tle 3. P. megkry soil inoulums lo effet on oo pos. Prmeter ss+pm ss+pm+ch ss+pm+ss nss nss+ch nss+ss Control D D D D D D D1 to D6: Dys fter inoultion. The ontrol is po inoulte with istille wter. Eh tretment ws repete twie. + = Presene of neroti lesion, - = Asene of neroti lesion. ss+pm: sterilize Soil + P. megkry; ss+pm+ch: sterilize Soil + P. megkry + Chitosn; ss+pm+ss: sterilize Soil + P. megkry + Snil Shell; nss: none sterilize Soil; nss + Chitosn: none sterilize Soil + Chitosn; nss + SS: none sterilize Soil + Snil Shell. nurseries n fiels in Cmeroon. Differenes tht emerge from see germintion, mesurement of plnt growth n quntittive proution of selete iohemil moleules, following the infetion of the plnts with P. megkry, provie numer of unerlying eviene to show tht soil tretment with orgni mtter oul improve growth of oo seelings n inue resistne to BPD. In our stuy, hitosn (up to 2%) n snil shell (up to 2%) see oting inrese the perentge of germintion ompre to ontrol. These results onfirm the erlier finings tht sees soke with hitosn inrese the germintion perentge (Zeng et l., 2012). This oul e ue to the exellent film-forming pity of hitosn, mking it esy to form semi-permele film on the see surfe whih n mintin the see moist n sor the moisture from the soil. Furthermore, Thereh et l. (2012) reporte tht sees ote with hitosn inrese the lipse n β-1,3 glunse enzyme tivity whih use etter germintion. However, espite the eviene of its tivity from mny stuies, the mehnism of hitosn effet on see germintion is still unknown. The plnt growth

9 Tyo et l. 339 promoting effets of hitosn oserve in this stuy re onsistent with the results of mny uthors who reporte the positive effets of hitosn inorporte into soil on erly growth stges of soyen, mini-tomto, upln rie n lettue (Hill et l., 2006). Like hitosn, the promoting effet of snil shell power on the growth of the oo seelings, is in greement with the stuy of Beuséjour et l. (2003) who showe tht soil menment with hitinous mteril stimulte grmpositive teri whih reue ommon s iniene n inue potto growth. The sene of neroti lesions on pos inoulte with soil trete with orgni mtter oul e ue to erese of P. megkry lo in the soil. This erese oul e orrelte to the helthier onition oserve in the trete plnts ompre to the ontrol plnts. Severl stuies hve lso reporte tht hitosn n hitinous soil menment, ontriute to plnt protetion y moifying the miroil ommunity n stimulting plnt efense mehnisms (Benhmou et l., 1994; Roy et l., 2010). Furthermore, Xing et l. (2013) showe tht nontrete n het-trete oyster shell power exhiite ntifungl tivities. This stuy shows tht the iniene of isese ws signifintly reue in plnts otine from soils trete with hitosn or snil shell. Uner ontrolle onitions, hitosn n snil shell were effiient eliitors of some efense retions in oo. This effet ws higher in plnts otine from soil trete with hitosn. The inution of systemi resistne oul e expline y the pity of hitosn n snil shell to stimulte plnt efense mehnisms n higher synthesis of plnt efense metolites like phenoli ompouns n pthogen-relte proteins (Benhmou n Pir, 2000; Mouo et l., 2010; Coqueiro et l., 2011). This inution of systemi resistne y snil shell oul e ue to its omposition. It is well known tht snil shell is minly ompose of lium ronte. Reently, Arfoui et l. (2015) emonstrte tht pretretment with lium se formultion enhne efense-relte genes expression in soyens response to Slerotini slerotiorum. This phenomenon ws onfirme in this stuy y higher proution of phenoli ompouns, totl ntive protein ontents n higher peroxise tivity in the leves of oo seelings following hitosn n snil shell soil tretment. At the onentrtion use, hitosn n snil shell oes not ffet the evelopment of the oo plnt. This effiy t low onentrtions suggests tht this ompoun ws reognize y plnt ells n the oserve protetion ws t lest prtly ue to the inution of plnt efense responses. In summry, this stuy ontriutes to show tht hitosn n snil shell soil tretment initite series of morphologil s well s iohemil hnges in the plnts whih re onsiere to e prt of the plnt efense response. The effet of snil shell on oo growth n resistne showe tht it is possile for snil shell power to e iofungiie n iofertilizer use in the ontrol of oo Blk Po Disese in nurseries. CONFLICT OF INTERESTS The uthors hve not elre ny onflit of interests. 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