Haustorium induction of parasitic plant: A new bioassay method to determine allelopathic potential

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1 /94 US $ 5.00 Allelopthy Journl 22 (2): (2008) Interntionl Allelopthy Foundtion 2008 Tle: 1, Figs : 3 Hustorium indution of prsiti plnt: A new iossy method to determine llelopthi potentil YIQING GUO 1, KIL-UNG KIM, IN-JUNG LEE, JOHN I. YODER 2 nd DONG-HYUN SHIN * Division of Plnt Biosienes, College of Agriulture & Life Sienes, Kyungpook Ntionl University, Degu , Repuli of Kore. E. Mil: dhshin@knu..kr (Reeived in revised form: July 25, 2008) ABSTRACT Allelopthy hs the potentil to redue the heriide use s omponent of integrted weed mngement tehnology. To develop new iossy method, we studied the effets of extrts from 3 rie ultivrs (Kouketsumohi, Dongjinyeo nd K21) on the hustorium indution rte, phytotoxiity nd ROS (retive oxygen speies) tivity of prsiti plnt Triphysri versiolor s the trget plnt. The three ultivrs influened the different rtes of hustorium indution. Kouketsumohi used the highest hustorium indution rte followed y K21 nd Dongjinyeo (lowest response t vrying onentrtions of rie tissue extrts). Applition of UV irrdition to the rie ultivrs inresed the phytotoxi effets on root growth of T. versiolor. Ativity of retive oxygen speies (ROS) ws ssyed y stining root tip of prsiti plnt with proe CM-H 2DCFDA [5-(nd 6)-hloromethyl-2, 7 - dihlorodihydro-fluoresein diette, etyl ester] nd monitoring the hnge of fluorresene. The fluoresene inresed when the dye ws oxidized y ROS. Fluoresene ppered in the shortest time in Kouketsumohi (llelopthi ultivr), followed y K21 nd Dongjinyeo (non-llelopthi ultivr). Results indited tht different hustorium rtes of rie ultivrs were relted to different ROS tivity. Thus T. versiolor n e used s new trget plnt to evlute the llelopthi potentil of plnt speies. Key words: Allelopthy, hustorium indution, prsiti plnt, rie, sreening method. INTRODUCTION Sustinle griulturl development requires finding eonomi nd eofriendly lterntives to onventionl weed ontrol methods, hene, mny ountries re spending on reserh nd tehnology development to meet the needs of sustinle griulture. The rie llelopthy my e one of the potent methods for sustinle griulturl system. Allelopthy is the effet of one plnt (inluding miroorgnisms) on growth of nother plnt through the relese of hemil ompounds into the environment (18). Allelopthy hs een widely studied world wide s evident from lrge numer of pulitions on different rops. Most of the llelopthy studies in griulture foussed on the prolems of ontinuous ropping, viz. utotoxiity nd effets on susequent rops (2,9,12,13,19,20,21). Rie llelopthy hs reeived gret ttention sine Dildy et l. (4,5) identified rie ultivrs exhiiting llelopthi potentil ginst duksld [Heternther * Correspondene uthor. 1 Yunnn Ademy of Agriulturl Sienes, Kunming , Chin. 2 Deprtment of Plnt Sienes, University of Cliforni-Dvis, Dvis, CA95616, USA.

2 372 Guo et l limos (Sw.) Wild] in the pddy field. Sine lte 1990s, Olofsdotter et l. (14-17) hve onduted tive reserh on rie llelopthy in the Interntionl Rie Reserh Institute (IRRI) nd pulished mny reserh ppers overing wider spets of rie llelopthy. The first step for llelopthy reserh is the development of proper sreening method. Although severl sreening methods re ville, these re not relted to rie llelopthy reserh. Kim nd Shin (11) stressed the need of universl iossy method, esy to rry out, relile nd eonomil to use in ll onditions, euse the sme rie ultivr shows different responses depending upon the sreening method used. Prsiti ngiosperms live in intimte ssoitions with their plnt hosts nd diretly ro them of wter nd nutrients. In some speies, host plnt is invded through hemil signling etween the host nd prsite. For exmple, prsiti Oronhee spp use moleules mde y the host root to trigger vrious developmentl proesses (seed germintion, host tthment nd invsion) nd forms the physiologil onduits through whih nutrients re trnsferred from host to prsite (6,10). Triphysri versiolor is fulttive generlist prsite, with rod host rnge i.e. monoots nd diots (1, 22) nd hs no griulturl signifine. The prsitism indued y other plnts provides n exeptionl opportunity to investigte the llelohemils relesed y plnts. Therefore, hustorium induing ftors nd phytotoxiity triggering ftors n e used to evlute the llelopthi potentil. We hve investigted the effets of extrts from 3 rie ultivrs (Kouketsumohi, Dongjinyeo nd K21) on the hustorium indution rte, phytotoxiity nd ROS (retive oxygen speies) tivity of T. versiolor. MATERIALS AND METHODS Prsiti plnt mteril: T. versiolor seeds were surfe-sterilized in 70% ethnol nd 2% NClO 2 for 15 min, followed y ddition of 1 drop of Tween 20 to the sterilizing medium. The seeds were rinsed fter 25 min, in 4 to 6 times volumes of sterile wter. Approximtely 200 seeds were pled in round petri dishes (90 mm 25 mm) ontining 0.1% Nole gr. The pltes were seled with prfilm nd kept t 4 C for 2 dys to stimulte germintion nd then trnsferred to growth inutor (16 C, 14 h light nd 10 h drk). Germintion ourred fter dys. The T. versiolor seedlings were gently removed from the germintion plte to the squre petri dish ontining 0.25x Hoglnd s medium, mde of mironutrients, 0.5% (w/v) surose, nd 1.0% (w/v) gr-c (Bio si In). Plnts were evenly sped in prllel rrngement ross the surfe of the medium. The roots of the prsiti plnt seedlings grew horizontlly in the medium. Pltes were seled with prfilm nd pled vertilly in growth hmer t C under light regime. Rie tissue extrts: Three test rie ultivrs: Kouketsumohi, Dongjinyeo nd K21, were grown septilly t 30 C under light regime for 2 weeks. Shoot nd root of 20-dysold- rie-seedlings were hrvested nd then ut into m lengths nd extrted in wter in rtio of 100 mg ml -1. The smples were pled in refrigertor overnight nd then filter-sterilized for further use. Hustorium indution in root tip: On Lminr flow, m long roots of

3 Biossy to determine llelopthi potentil 373 T. versiolor seedlings in the petri dishes, were seleted nd used for hustorium indution. Indution ws hieved y treting the root tips of the prsiti seedlings with the filtersterilized rie tissue extrts nd susequently, plted. The pltes were pled in the growth hmer. The root tips were heked t the given time under disseting mirosope t x mgnifition. The plnts were sored s indued/responders (with hustori/root hirs) nd un-indued/non- responders (without hustori/root hirs) nd then hustorium indution rte were determined fter 60 h. Phytotoxi tivity: Extrts from rie tissue were pplied to the roots of T. versiolor seedlings s desried for the hustorium iossys. Phytotoxi tivity of the extrts ws determined y mesuring the root growth of T. versiolor 60 h fter tretment. Mesurement of ROS: ROS (retive oxygen speies) ws ssyed y stining root tip of prsiti plnts with proe CM-H 2 DCFDA [5-(nd 6)-hloromethyl- 2,7 -dihlorodihydrofluoresein diette, etyl ester] nd monitoring the hnge of fluoresene (3). An inrese in fluoresene ws oserved when the dye ws oxidized y ROS to dihlorofluoresein (DCF). Response of trget plnts to extrts from rie tissue: Prsiti T. versiolor, rnyrd grss nd lettue were used s trget plnts. Extrts from rie tissue ws pplied to the root tip of trget plnts with m length of root. The effet of the extrts ws determined y mesuring the root length 48 h fter tretment for rnyrd grss nd lettue, nd fter 60 h for the prsiti plnt. RESULTS AND DISCUSSION Hustorium indution Extrts of oth rie shoot nd root indued the hustorium in T. versiolor in ll rie ultivrs. Twelve to 24 h fter the rie extrt tretment, the tip of T. versiolor root swelled (Fig. 1). Among the different ultivrs, higher onentrtion (10%) of the shoot extrts gve no signifint differenes in hustorium indution ompred to lower onentrtions (<5%), whih produed signifint effets. This showed tht the rte of hustorium indution mong extrts of the vrious ultivrs depended on the onentrtion of the extrts. As the onentrtion of extrts deresed, hustorium induing rte lso deresed. The pplition of UV irrdition gve higher hustorium induing rte thn the non-uv irrdition (Fig. 2). Root extrts hd similr effets on hustorium indution, ut the rtes were lower thn those of shoot extrts t ll onentrtions (Fig. 2). The results suggested tht ll the three rie ultivrs hd hustorium induing ftor nd ould serve s host plnts. In ddition to this, UV stress my indue prodution of more llelohemils s hustorium induing ftor in plnts. Phytotoxi effets Root growth of prsiti plnt ws grdully inhiited s the onentrtion of rie extrts inresed. Kouketsumohi, n llelopthi ultivr, showed more inhiitory effet thn Dongjinyeo, non-llelopthi ultivr nd their progeny K21, showed the

4 374 Guo et l Dongjinyeo K21 Kouketsumohi 12h 24h Time fter tretment Figure 1. Time ourse of hustorium formed s shoot extrts of 5% from different rie ultivrs were treted to the root tips of T. versiolor.

5 Biossy to determine llelopthi potentil 375 inhiitory effet etween its prents (Fig. 3-E). The higher phytotoxi effets were oserved in UV-irrdited ultivrs thn in the non-uv-irrdited ones. The greter inhiitory effet in response to UV-irrdition ws otined from the individul plnts with the higher hustorium indution rte. This suggested tht there ws lose reltion etween the phytoxiity nd hustorium induing ftors with respet to the llelohemils. Prodution of ROS Plnt ells produe retive oxygen speies (ROS), whih might e ffeted y environmentl stresses. To determine ROS, CM-H 2 DCFDA, nmed 5-(nd 6)- hloromethyl-2,7 -dihlorodihydrofluoresein diette, etyl ester, ws pplied to the

6 376 Guo et l Extrt onentrtion (%) Extrt onentrtion (%) Extrt onentrtion (%) Extrt onentrtion (%) Figure 2. Effets of rie extrts on hustorium indution rte. (A) Shoot extrt, (B) Shoot extrts fter UV-irrdition, (C) Root extrts, (D) Root extrts fter UV- irrdition. Dt re mens of three independent experiments with 7-10 plnts for eh determintion. The vertil rs represent men ± stndrd error nd sme letter indite no signifint differene t 5% level (DMRT). root tip. After tretment with rie shoot extrts for 2 h, 25 µm of proe ws dded to the root tip of T. versiolor nd the fluoresene prodution immeditely oserved. At higher rie extrt onentrtion (10%), it took longer time to oserve the fluoresene from the root tip. However, time for the fluoresene ourrene ws shortened t 5% onentrtion of the extrts. UV irrdition promoted the hnge of dye proe y induing rpid prodution of llelohemils in the sme ultivr (Tle 1). Kouketsumohi nd K21 hd shorter response time to the proe thn Dongjinyeo t 5%

7 Biossy to determine llelopthi potentil 377 Extrt onentrtion (%) Extrt onentrtion (%) Extrt onentrtion (%) Extrt onentrtion (%) Extrt onentrtion (%) Extrt onentrtion (%) Figure 3. Effets of rie shoot extrts on root growth of trget plnts. (A) Brnyrd grss treted y rie shoot extrts, (B) UV-irrdited rie shoot extrts, (C) Lettue treted y rie shoot extrts, (D) UVirrdited rie shoot extrts, (E) T. versiolor treted y rie shoot extrts, (F) UV-irrdited rie shoot extrts. The vertil rs represent men ± stndrd error nd sme letters indite no signifint differene t 5 % level y DMRT.

8 378 Guo et l onentrtion of the extrts, whih mthed the llelopthi potentil mong the ultivrs. At higher onentrtion of 10%, the metoli proess my e inhiited, so tht slow response ws oserved to the dye proe. Tle 1. Time ourse of ourrene of DCF fluoresene fter the root tips of T. versiolor were treted with rie extrts Rie ultivrs Shoot extrts onentrtion (%) 5% 10% Non-UV UV-irrdition Non-UV Kouketsumohi * K Dongjinyeo *Time unit: seond. Dt with the sme letter within olumns re not signifintly different (DMRT, P 0.05). Rie ultivrs The root tips of trget plnts were treted with the rie extrts nd root growth ws determined y mesuring their root length 60 h fter tretment for T. versiolor nd 48 h for lettue nd rnyrd grss. As the onentrtion of the extrts derese from 10% to 2.5%, phytotoxi effet lso deresed (Fig 3). UV irrdition resulted in higher inhiitory effet in ll the three ultivrs. At 10% onentrtion, Kouketsumohi showed the highest inhiitory effets on rnyrd grss nd Dongjinyeo. The lowest inhiitory effets on lettue were lso oserved in Dongjinyeo. Kouketsumohi nd K21 hd higher inhiitory effets on the root growth of T. versiolor thn Dongjinyeo. Thus the response of trget plnts to shoot extrts of rie ultivrs seemed to e onsistent with llelopthi potentil of rie ultivrs. The fulttive prsite, Triphysri versiolor, develops hustorium only in the presene of host ftors. An in vitro ssy identified severl phenoli derivtives tht trigger hustorium formtion when pplied to Triphysri roots inluding simple phenolis, flvonoids nd quinones (1). It ws suggested tht prsiti plnts reognize llelohemils derived from host plnts. From the oservtion of hustori indution rte, it ws ler tht the three rie ultivrs hd hustorium induing ftor. It is known tht prsiti plnts use quinines to initite the development of hustorium. DMBQ is n tive induer of Triphysri hustorium etween 1 nd 30 μm onentrtions. At onentrtions of 100 μm or higher it is phytotoxi nd Triphysri roots turn rown nd die (8). Quinones nd phenolis re most ommon lsses of llelopthi phytotoxins (7). Quinones oxidize phenols nd phenols redue quinones, nd eletril trnsformtions etween them re of muh iologil signifine. From the oservtions, hustorium indution of prsiti plnt, phytotoxi tivity nd ROS prodution were relted to llelopthi potentil in rie. In onlusion, oth tissue extrts n hve either hustorium induing or phytotoxi effets depending on their onentrtions. There is lose reltionship etween phytotoxi tivity nd hustorium indution ftor. It seems tht hustorium induing ftors nd phototoxi tivity re struturlly relted to phenoli ompounds.

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