TESTING EFFECTS OF MYCORRHIZAL FUNGI ON GROWTH AND DEVELOPMENT OF ABRONIA MACROCARPA THESIS

Transcription

1 TESTING EFFECTS OF MYCORRHIZAL FUNGI ON GROWTH AND DEVELOPMENT OF ABRONIA MACROCARPA THESIS Presented to the Grdute Council of Texs Stte University-Sn Mrcos in Prtil Fulfillment of the Requirements for the Degree Mster of SCIENCE by Stefnie Ferrzzno, B.S. Sn Mrcos, Texs August 2012

2 TESTING EFFECTS OF MYCORRHIZAL FUNGI ON GROWTH AND DEVELOPMENT OF ABRONIA MACROCARPA Committee Members Approved: Pul S. Willimson, Chir Tin M. Cde Thoms R. Simpson Approved: J. Michel Willoughby Den of the Grdute College

3 COPYRIGHT by Stefnie Ferrzzno 2012

4 FAIR USE AND AUTHOR S PREMISSION STATEMENT Fir Use This work is protected by the Copyright Lws of the United Sttes (Public Lw , section 107). Consistent with fir use s defined in the Copyright Lws, brief quottions from this mteril re llowed with proper cknowledgement. Use of this mteril for finncil gin without the uthor s express written permission is not llowed. Dupliction Permission As the copyright holder of this work I, Stefnie Ferrzzno, refuse permission to copy in excess of the Fir Use exemption without my written permission.

5 ACKNOWLEDGMENTS I would like to thnk my dvisor nd committee chir, Dr. Pul S. Willimson nd my committee members, Dr. Tin M. Cde nd Dr. Thoms R. Simpson. I would lso like to thnk Luren Cody, Joey Escmill, Jcki M, nd Flo Oxley for their help in the field, Dr. G. Upchurch for letting me use his fume hood, Nimmi Krunrthn for helping tke microgrphs, nd Adm Durte for his dvice on sttisticl grphics. Thnk you to my fmily nd friends for their ongoing support. Finlly, specil thnks to Mycorrhizl Applictions, Inc. for their generous dontion of inoculnt for the purposes of this study. This mnuscript ws submitted on June 12, v

6 TABLE OF CONTENTS Pge ACKNOWLEDGMENTS...v LIST OF FIGURES... vii ABSTRACT... ix CHAPTER I. INTRODUCTION...1 II. MATERIALS AND METHODS...6 Study Species... 6 Mycorrhizl Inocultion Experiment...7 Exmintion of Nturlly Occurring Mycorrhize...9 III. RESULTS...13 Mycorrhizl Inocultion Experiment...13 Exmintion of Nturlly Occurring Mycorrhize...24 IV. DISCUSSION...27 LITERATURE CITED...32 vi

7 LIST OF FIGURES Figure Pge 1. Germintion percentges s of Mrch 2011 due to trnsect Germintion percentges s of Mrch 2011 due to tretment Men percent survivorship from Mrch of 2011 to Mrch of 2012 for control nd tretment ctegories Totl number of plnts in ech developmentl stge for ech tretment ctegory in Mrch of Totl number of plnts in ech developmentl stge for ech tretment ctegory in April of Totl number of plnts in ech developmentl stge for ech tretment ctegory in Mrch of Totl number of plnts in ech developmentl stge for ech tretment ctegory in April of Men number of leves per plnt in control nd tretment plots in Mrch of Men number of leves per plnt in ech of the experimentl trnsects in Mrch of Men number of leves per plnt in control nd tretment plots in April of Men number of leves per plnt in ech of the experimentl trnsects in April of vii

8 12. Men eril dimeter of plnts in control nd tretment plots in April of Men number of leves per plnt in control nd tretment plots in Mrch of Men eril dimeter of plnts in control nd tretment plots in Mrch of Men height of plnts in control nd tretment plots in Mrch of Men number of leves per plnt in control nd tretment plots in April of Men eril dimeter of plnts in control nd tretment plots in April of Men height of plnts in control nd tretment plots in April of Lentil root smples with observble AMF coloniztion Nturlly occurring mycorrhize fter stining with Sheffer blck ink (non-wterproof)...26 viii

9 ABSTRACT TESTING EFFECTS OF MYCORRHIZAL FUNGI ON GROWTH AND DEVELOPMENT OF ABRONIA MACROCARPA by Stefnie Ferrzzno, B.S. Texs Stte University-Sn Mrcos August 2012 SUPERVISING PROFESSOR: PAULA WILLIAMSON Endngered nd thretened species require vrious mngement plns for recovery, mny of which include reintroductions. Abroni mcrocrp, n endngered Texs endemic plnt species, hs been suggested s potentil cndidte for reintroduction. Inocultion with mycorrhizl fungi hs been component of reintroduction plns for some species. Mycorrhizl fungi hve mutulistic, obligtory symbiotic reltionship with most higher-order plnts. They cn increse their host s uptke of nutrients such s P, N, nd K, increse plnt growth, reduce sline nd lkline toxicity, nd increse drought resistnce. The effects of mycorrhize on A. mcrocrp hd not been studied. I hypothesized tht growth nd development of A. mcrocrp would be incresed when plnts were inoculted with mycorrhizl fungi. I estblished 3 ix

10 trnsects on privte property in Freestone County, TX with 6 plots ech. Hlf of the plots were rndomly selected to be plnted with inoculted seed while the other hlf were plnted with seed coted with utoclved inoculnt s control. I collected dt for two yers nd nlyzed mesurements of growth nd development. Results indicted tht growth ws significntly improved by inocultion in the first yer fter germintion. Men number of leves per plnt ws greter in tretment plots in Mrch 2011 (P = ), nd men eril dimeter of plnts in tretment plots ws lrger in April 2011 (P = 0.018). Plnts in tretment plots were lso lrger in eril dimeter nd height in the second yer of growth. However, these differences were not sttisticlly significnt. Germintion, survivorship, nd development were not ffected by tretment, but there ws some observble vrition in germintion due to trnsect. This suggests tht A. mcrocrp is extremely sensitive to vritions in microhbitt. Positive results in the first yer of growth wrrnt further study of mycorrhizl interction with this species. x

11 CHAPTER I INTRODUCTION Our plnet is presently undergoing one of the lrgest extinction events the world hs ever known. Extensive hbitt loss nd degrdtion re contributing to species extinctions occurring t n lrming rte, resulting in rpid loss of overll biodiversity (Novcek nd Clelnd, 2001). As result, mny extnt species of nimls nd plnts re thretened or endngered. Humn ctivities, primrily conversion of nturl hbitt into griculturl, urbn, nd recretionl res re the gretest current threts to biodiversity (Sebloom et l., 2002). Ironiclly, humn intervention my be the only mens of mintining, or possibly recovering biot in the fce of this environmentl ctstrophe (Novcek nd Clelnd, 2001). Active mngement strtegies such s hbitt restortions, rtificil outplnting, nd reintroductions tht might estblish popultions in historiclly pproprite hbitts re often necessry in these situtions (Fisher nd Jychndrn, 2002). Reintroduction procedures hve become commonplce over the pst two decdes. As of 1992, nerly one-fourth of the plnts listed under the Endngered Species Act hve recovery plns tht include reintroduction progrms (Flk nd Olwell, 1992). For exmple, the recovery pln for Abroni mcrocrp, the lrge-fruited snd-verben, includes reintroduction s potentil mens of downlisting or delisting the txon. Abroni mcrocrp is Texs 1

12 2 endemic plnt species listed s federlly endngered on September 28, 1988 (U.S. Fish nd Wildlife Service, 1988) nd endngered in Texs on December 30, 1988 (U.S. Fish nd Wildlife Service, 1992). The U.S. Fish nd Wildlife Service designted recovery priority of 2 for the species, which denotes high degree of thret, but gret potentil for recovery (U.S. Fish nd Wildlife Service, 1992). The recovery pln for this species requires t lest 20 vible nd persistent popultions, ech of t lest hectres (25 cres) in size with popultion of t lest 600 individuls, before it cn be delisted (U.S. Fish nd Wildlife Service, 1992). There re currently nine known A. mcrocrp popultions confined to three Texs counties (Leon, Robertson, nd Freestone); ll popultions occur on privtely owned property. Eleven new popultions of A. mcrocrp must be discovered or creted in order to meet the recovery gol. If 11 nturlly occurring popultions re not identified, reintroduction will be criticl to recovery. In order to mximize the chnces of successful reintroductions, biologists must first become knowledgeble bout the optiml conditions required by given species for it to estblish nd reproduce. Much of the informtion required to develop reintroduction pln exists for A. mcrocrp; therefore, this species should be considered prime cndidte for reintroduction. Studies hve been conducted on phenology (Willimson et l., 1994), reproductive biology (Willimson et l., 1994; Willimson nd Bzeer, 1997), popultion genetics (Willimson nd Werth, 1999), popultion structure nd hbitt chrcteristics (Meredith, 2006) nd seed germintion (Goodson nd Willimson, 2011). If biologists hope to recover A. mcrocrp, it is impertive tht reserchers continue to dd to the growing librry of knowledge concerning this endngered species.

13 3 In order for successful reintroduction to occur, specific set of conditions must be met tht mimic the chrcteristics of nturlly occurring popultion (Pvlik, 1996). These hbitt conditions include the nutrient nd biot content of soils in the re. Soils t nturlly occurring popultions of A. mcrocrp re low in nitrtes (2-11 ppm), phosphorus (13-29 ppm), nd potssium (24-39 ppm) (Meredith, 2006). A pilot reintroduction study by Willimson (2008) found tht A. mcrocrp hd n incresed rte of growth nd development when plnted in soils tht were rich in potssium (81 ppm) nd contined the upper levels of nitrogen (15 ppm) nd phosphorus (29 ppm) known in soils supporting popultions of A. mcrocrp. In nturlly occurring popultions, plnts remin in the seedling stge the first yer fter germintion nd do not rech juvenile nd reproductive stges for nother one to two yers. However, seeds plnted in these nutrient-rich soils sometimes reched the juvenile or even reproductive stges within the first yer fter plnting (Willimson, 2008). One possible method for chieving enriched nutrient conditions is through the presence of mycorrhizl fungi. It is known tht there is n existing reltionship between A. mcrocrp nd rbusculr mycorrhizl fungi (AMF) in nturlly occurring conditions (Willimson, unpublished dt). However, it is not known if there is the potentil to crete one when reintroducing the species. The effect of AMF on growth nd development of the species is lso not known. Arbusculr mycorrhizl fungi hve mutulistic, obligtory symbiotic reltionship with most higher-order plnts (White et l., 2008). The fungl myceli extend both into the roots of the host plnt nd into the surrounding soil. Inorgnic compounds found in the substrte cn thus flow from fungus to plnt, while orgnic

14 4 compounds flow from plnt to fungus (Allen, 1991). Plnts treted with chemicl fungicides often suffer s result of the deth or reduction of their mycorrhizl popultions nd hve to be supplemented with mycorrhizl inoculnts in order to recover (Plenchette et l., 1983). Mny lbortory nd field studies hve shown tht plnts receive vriety of benefits from AMF. These often include improved nutrient bsorption nd greter tolernce of drought (Smith nd Red, 1997). A study on Arucri ngustifoli showed tht inocultion with AMF gretly improved the growth of this plnt, incresing the mount of shoot biomss in comprison to root biomss (Zndvlli et l., 2004). Plnts in sline nd lkline soils inoculted with AMF hve the potentil to increse their survivl, growth, nd reproduction (Zhng et l., 2011). The sme study showed tht ssocitions with AMF led to selective nutrient bsorption. Uptke of N, P, C, nd K were incresed, nd bsorption of N nd Cl were reduced. This indicted tht mycorrhizl ssocition hd the potentil to optimize nutrient content in host plnts. These ttributes should led to n incresed rte of growth nd development for plnts with AMF, even in soils with reltively low nutrient concentrtions. Propgules of mycorrhizl fungi tht hve the potentil to initite coloniztion include spores, infected root frgments, nd myceli (Hrley nd Smith, 1983). Inocultions with AMF cn be used to reestblish plnt communities tht hve been degrded for resons such s soil disturbnce nd erosion (Zhng et l., 2011). Severl studies hve investigted the usefulness of AMF tretments to enhnce the performnce of highly thretened or endngered plnts nd found tht the use of AMF hs positive effects on nutrient uptke, growth, nd survivl (Brroetven et l., 1998; Fisher nd

15 5 Jychndrn, 2002; Pnwr nd Vys, 2002; Zubek et l., 2009). These studies collectively demonstrte tht AMF cn benefit wide vriety of plnt species, nd tht its use cn improve the success of reestblishment efforts. Evidence lso suggests tht the origin nd species of the microbiot used for inocultion re often of little importnce. Whether the inoculnt is isolted from the plnt s nturl environment or purchsed from commercil source, the observble benefits re similr (White et l., 2008; Zubek et l., 2009). The primry objective of this study ws to determine if A. mcrocrp would benefit from AMF inocultion under field conditions. I hypothesized tht inocultion with mycorrhizl fungi should increse nutrient uptke in A. mcrocrp, resulting in incresed rtes of growth nd development, even in low-nutrient soils. If A. mcrocrp showed incresed growth nd rte of development when inoculted with AMF, inocultion could be used s reintroduction tool in recovery of this species.

16 CHAPTER II MATERIALS AND METHODS Study Species Abroni mcrocrp, commonly known s the lrge-fruited snd-verben, ws first described by Gllowy in It is not true verben, but member of the Nyctgincee fmily which is lso referred to s the four o clock fmily becuse the flowers of its plnts open t round 3:00 p.m. to 4:00 p.m (Gllowy, 1972). Abroni mcrocrp blooms from Februry to June, with pek flowering in Mrch nd April, nd produces inflorescences composed of 27 to 40 flowers ech (Willimson et l., 1994). They rnge from light pink to fuchsi in color nd re tubulr in shpe, growing up to 3.2 centimeters in length. This species is n herbceous perennil tht grows up to 20 centimeters in height nd hs lrge tproot. The leves re ovl-shped, covered with glndulr hirs, nd re oppositely rrnged long the stem. The fruit produced by A. mcrocrp is n nthocrp. This consists of dry, ppery outer portion formed by the lower clyx which encses n chene. Achenes re dry, indehiscent, single-seeded fruits tht re unttched to the pericrp. Interestingly, the ppery nthocrp develops whether the flower hs been pollinted or not. However, in the bsence of pollintion, the chene within fils to develop (Willimson et l., 1994). This species relies solely on pollintors for fruit set becuse it is incpble of successfully 6

17 7 self-fertilizing (Willimson nd Bzeer, 1997). Common pollintors of this species include hwk moths (Sphingide) nd noctuid moths (Noctuide) (Willimson et l., 1994). Anthocrps of A. mcrocrp re wind-dispersed, but the mjority fll within 30 centimeters of the prent plnt (Willimson nd Werth, 1999). Spcil distribution in nturlly occurring popultions is therefore described s clumped-contgious, mening tht if there is n individul of the species present, there is high probbility tht others re nerby (Willimson nd Werth, 1999). This distribution pttern my lso be contributing fctor to the txon s rrity nd smll rnge size. Mycorrhizl Inocultion Experiment The mycorrhizl inocultion experiment ws conducted on privte property in Freestone County, Texs nd ws chosen bsed on its potentil to support popultions of A. mcrocrp bsed on edphic fetures nd community composition. Lndowner permission to conduct this experiment ws obtined. Abroni mcrocrp seeds were collected from n existing popultion t Hilltop Lkes (Leon Co., Texs). At the study site, I estblished three trnsects, ech mesuring 30 m in length nd contining six plots ech. I plced ech trnsect t lest 12 m wy from the others. The plots mesured 1 m 2 nd were seprted by buffer zone of 4 meters in order to void indvertent coloniztion of AMF in the control plots due to the spreding of mycorrhize from the inoculnt. I then rndomly ssigned ech plot to one of two tretments. Experimentlly treted plots were plnted with A. mcrocrp seed tht hd been coted with n AMF inoculnt in powder form. The other hlf (control plots) were plnted with seeds tht I treted with n inoculnt which hd been utoclved to kill the mycorrhize. The AMF

18 8 inoculnt ws supplied by Mycorrhizl Applictions, Inc. nd contined four species of mycorrhizl fungi: Glomus intrrdices, Glomus mossee, Glomus ggregtum, nd Glomus etunictum. The inoculnt contined 220 propgules/g. I plnted seventy-five A. mcrocrp seeds in ech plot in the spring of I lso pplied supplementl dose of inoculnt to the tretment plots in the spring of 2011 to ensure tht the mycorrhize mde contct with roots. Ech plnt in the experimentl plots received 2 ml of suspension contining 10% mycorrhizl inoculnt nd 90% DI wter. I dministered 2 ml of plin deionized wter to plnts in control plots. In Mrch of 2011, I identified individuls of A. mcrocrp tht hd successfully germinted nd clculted percent germintion for tretment nd control plots. I lso mrked plnts numericlly to keep trck of individuls in ech plot. However, I ws unble to consistently trck individuls becuse of multiple soil disturbnces cused by nimls t the field site, most likely ferl hogs. Abroni mcrocrp only grows in height from lte Februry to lte April or erly My. After this time, the bove-ground prt of the plnt dies bck, leving only the tproot nd shoot pex buried in the soil (Willimson nd Bzeer, 1997). For this reson, I only recorded mesurements of growth in the spring months. Since A. mcrocrp is endngered, it ws importnt not to destroy the resulting plnts. Therefore, I used severl proxies to mesure growth in lieu of mesuring dry biomss of the plnts. In Mrch of 2011, I recorded number of leves, developmentl stge, nd number of inflorescences present. Developmentl stge ws clssified s seedling, juvenile, or t nthesis. I clssified plnts s seedlings if they hd only emerged cotyledons, s juveniles if they hd three or more leves but no inflorescences, nd t nthesis if

19 9 inflorescences were present. In April 2011, Mrch 2012, nd April 2012, I recorded number of leves, eril dimeter, height, developmentl stge (seedling, juvenile, t nthesis), nd number of inflorescences present. I lso clculted percent survivorship from spring of 2011 to spring of 2012 for tretment nd control plots. I nlyzed percent germintion using two-fctor ANOVA with the experimentl tretment s one fctor nd individul trnsects s the other in order to determine if there ws sttisticlly significnt influence due to differences in loction in ddition to the presence or bsence of AMF inocultion. I used seprte two-fctor ANOVA to nlyze percent survivorship from Mrch 2011 to Mrch I conducted seprte Chi-Squred tests of independence on developmentl stge dt collected in Mrch nd April Frequencies of plnts t ech developmentl stge were often fewer thn 5 per tretment regime, so Fisher s Exct tests were used for dt collected in Mrch nd April For lef count dt collected in Mrch 2011, I used three-fctor ANOVA with tretment nd trnsect crossed nd plot nested within trnsect. For ll other dt sets, I used seprte multifctoril MANOVAs for nlysis of growth. Once gin, tretment nd trnsect were crossed while plot ws nested within trnsect. Univrite equivlent nlyses were conducted when significnt results were found in MANOVAs. All sttisticl nlyses were performed using R softwre version (R Development Core Tem, 2009).

20 10 Exmintion of Nturlly Occurring Mycorrhize Root smples from ssocited plnts in the re were screened during spring 2011 to determine if mycorrhizl ssocition lredy existed in the re. The stining technique I used ws implemented following procedures from Vierheilig et l. (1998). The procedure employs non-toxic stin composed of mixture of regulr fountin pen ink nd white household vinegr. However not ll brnds of ink re functionl in this tsk (Vierheilig et l., 1998). In ddition, most ink brnds used in the study performed by Vierheilig et l. (1998) re not redily vilble in the United Sttes. For these resons, it is recommended tht pressy be conducted with severl different inks. I conducted the pressy using roots of green lentil plnts (Lens culinris). The inks used were Speedbll Super Blck Indi Ink (wterproof), Bomby Blck Ink (wterproof), Sheffer Blck ink (non-wterproof), Higgins Blck Ink (wterproof), nd Higgins Blck Ink (non-wterproof). All were chosen bsed on their vilbility in the United Sttes. I plnted 50 green lentil seeds in soil inoculted with the sme mycorrhizl formul used to tret seeds of A. mcrocrp in the field study. To ccomplish this, I plced regulr potting soil in plnting trys nd mde indenttions for ech seed before sprinkling 5 g of inoculnt in ech cvity. I then plced one green lentil seed in ech cvity nd covered it with pproximtely 1 cm of soil. I kept the resulting plnts on dy/night cycle of 16h/8h with constnt temperture of 25 0 C nd hrvested them 8 weeks fter sprouting. I rinsed the roots in tp wter to remove soil nd debris nd seprted them from the rest of the plnt. I cut the smples into segments mesuring 2 cm in length nd soked them in wter overnight.

21 11 I submerged the root segments in boiling queous 10% KOH solution until clered before rinsing them repetedly with tp wter. I eqully divided the clered segments mong the five ink tretments nd boiled ech segment group for 5 min in 5% ink-vinegr solution comprised of one of five brnds of blck writing ink (Speedbll Super Blck Indi Ink, Bomby Blck Ink, Sheffer Blck ink, Higgins Blck Ink wterproof, nd Higgins Blck Ink non-wterproof) nd plin white vinegr contining 5% cetic cid. I then rinsed the smples severl times with tp wter slightly cidified with vinegr. I observed ech segment under compound microscope nd scored it for presence/bsence of mycorrhizl infection in order to clculte percent infection for ech smple group. If there ws ny visible trce of mycorrhizl infection in given root segment, I scored it s present, nd if there ws no visible infection in given root segment, I scored it s bsent. I compred percent infection for ech of the smple groups to determine if there ws sttisticlly significnt difference in the mount of observed root coloniztion due to ink brnd nd used 95% Confidence Intervl to clculte error for ech smple group. I ssessed the usefulness of ech ink brnd bsed on the bility to stin AMF nd the bility to differentite between stined fungl cells nd the surrounding plnt tissue. I used the ink brnd determined to be most suitble bsed on this pressy to test ntive plnts collected from the field site for presence nd extent of AMF infection. Texs sndmint (Rhododon cilitus), silver croton (Croton rgyrnthemus), nd the common plntin (Plntego mjor) re plnts commonly ssocited with suitble A. mcrocrp hbitt (Meredith, 2006). I collected t lest 10 smples from ech of these three species, nd screened their roots for AMF using the forementioned procedure with

22 12 the most suitble ink. I then clculted percent infection nd error using 95% Confidence Intervl for ech ssocited plnt species nd used these results to help determine the degree of AMF coloniztion lredy present t the field site excluding experimentl tretments.

23 CHAPTER III RESULTS Mycorrhizl Inocultion Experiment Totl germintion for control plnts ws 16.19% while totl germintion for plnts treted with AMF inoculnt ws 17.63% (Fig. 1). A two-fctor ANOVA indicted tht there ws no significnt difference in percent germintion due to tretment (F = , df = 1, P = ), but there ws difference due to trnsects (F = , df = 2, P = ). Trnsect 1 hd men germintion of 13.33%, trnsect 2 hd men germintion of 12.67%, nd trnsect 3 hd men germintion of 24.73% (Fig. 2). There ws lso no significnt difference in survivorship between tretments ccording to twofctor ANOVA (F = , df = 1, P = ). From Mrch 2011 to Mrch 2012, totl control plnt survivl ws 14.67%, nd totl inoculted plnt survivl ws 17.0% (Fig 3). Developmentl stge ws clssified s seedling, juvenile, or t nthesis. A Chi- Squred test of independence for developmentl stge of plnts in Mrch of 2011 reveled tht developmentl stge of plnts ws not dependent on tretment (X 2 = , df = 1, P = ) (Fig. 4). Developmentl stges in April of 2011 were lso independent of tretment (X 2 = , df = 1, P = ) (Fig. 5). Fisher s Exct tests on developmentl stge dt collected in Mrch (P = 1.0) (Fig. 6) nd April of 2012 (P = 13

24 Percent Germintion Percent Germintion ) (Fig. 7) lso reveled no dependence on tretment. Throughout the experiment, only one plnt reched nthesis b 13.33% 12.67% 24.73% Trnsects Figure 1. Germintion percentges s of Mrch 2011 due to trnsect. (F = , df = 2, P = ). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different % 17.63% Control Tretment Experimentl Tretment Figure 2. Germintion percentges s of Mrch 2011 due to tretment. (F = , df = 1, P = ). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

25 Number of Plnts Percent survivorship % 17.00% Control Tretment Experimentl Tretment Figure 3. Men percent survivorship from Mrch of 2011 to Mrch of 2012 for control nd tretment ctegories. Effect due to tretment (F = , df = 1, P = ). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different Seedling Juvenile Anthesis Developmentl Stge Control Tretment Figure 4. Totl number of plnts in ech developmentl stge for ech tretment ctegory in Mrch of (X 2 = , df = 1, P = ).

26 Number of Plnts Number of Plnts Number of Plnts Seedling Juvenile Anthesis Developmentl Stge Control Tretment Figure 5. Totl number of plnts in ech developmentl stge for ech tretment ctegory in April of (X 2 = , df = 1, P = ) Seedling Juvenile Anthesis Developmentl Stge Control Tretment Figure 6. Totl number of plnts in ech developmentl stge for ech tretment ctegory in Mrch of (P = 1.0) Seedling Juvenile Anthesis Developmentl Stge Control Tretment Figure 7. Totl number of plnts in ech developmentl stge for ech tretment ctegory in April of (P = ).

27 Men Number of Leves Men Number of Leves 17 Anlysis of lef count dt collected in Mrch 2011 showed tht there were significnt differences in lef numbers of plnts due to tretment (F = , df = 1, P = ) with inoculted plnts hving higher lef counts (Figure 8). Lef counts lso differed significntly mong trnsects (F = , df = 2,P = 2.8x10-13 ) nd plots (F = 2.348, df = 14, P = ). Individuls in trnsect 1 verged leves per plnt, those in trnsect 2 verged leves per plnt, nd those in trnsect 3 verged 2.82 leves per plnt (Figure 9) b Control Tretment Experimentl Tretment Figure 8. Men number of leves per plnt in control nd tretment plots in Mrch of (F = , df = 1, P = ). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different b Trnsect Figure 9. Men number of leves per plnt in ech of the experimentl trnsects in Mrch of (F = , df = 2, P = 2.8x10-13 ). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

28 Men Number of Leves 18 MANOVA nlysis of growth dt collected in April 2011 showed significnt effects due to tretment (Pilli Trce = , P = ), trnsect (Pilli Trce = , P = 2.287x10-5 ), nd plot (Pilli Trce = , P = 0.021). I therefore conducted equivlent ANOVAs for both lef count nd eril dimeter of plnts. These reveled tht lef count ws not significntly influenced by tretment (F = , df = 1, P = ) but ws significntly influenced by trnsect (F = 4.224, df = 2, P = 0.014), while eril dimeters of plnts were significntly influenced by tretment (F = 5.707, df = 1, P = 0.018) nd plot (F = , df = 14, P = ), with inoculted plnts hving lrger eril dimeters (Figs. 10, 11, 12) Control Tretment Experimentl Tretment Figure 10. Men number of leves per plnt in control nd tretment plots in April of (F = , df = 1, P = ). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

29 Men Aeril Dimeter (cm) Men Number of Leves b Trnsect Figure 11. Men number of leves per plnt in ech of the experimentl trnsects in April of (F = 4.224, df = 2, P = 0.014). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different b Control Tretment Experimentl Tretment Figure 12. Men eril dimeter of plnts in control nd tretment plots in April of (F = 5.707, df = 1, P = 0.018). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

30 Number of Leves 20 Anlysis of growth dt collected in Mrch 2012 showed tht lef count (Fig. 13), eril dimeter of plnts (Fig. 14), plnt height (Fig. 15), nd number of inflorescences were ll not significntly different between plnts in tretment nd control plots (Pilli Trce = , P = 0.36), mong trnsects (Pilli Trce = , P = 0.149), or mong plot (Pilli Trce = , P = 0.817). Since there ws only one plnt to rech nthesis, the experiment-wide totl number of inflorescences ws 2. Growth dt collected in April 2012 lso yielded insignificnt differences due to tretment (Pilli Trce = , P = 0.51), trnsect (Pilli Trce = , P = 0.099), nd plot (Pilli Trce = , P = 0.69) (Figs. 16, 17, 18) Control Tretment Experimentl Tretment Figure 13. Men number of leves per plnt in control nd tretment plots in Mrch of (Pilli Trce = , P = 0.36). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

31 Men Height (cm) Men Dimetier (cm) Control Tretment Experimentl Tretment Figure 14. Men eril dimeter of plnts in control nd tretment plots in Mrch of (Pilli Trce = , P = 0.36). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different Control Tretment Experimentl Tretment Figure 15. Men height of plnts in control nd tretment plots in Mrch of (Pilli Trce = , P = 0.36). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

32 Men Aeril Dimeter (cm) Men Number of Leves Control Tretment Experimentl Tretment Figure 16. Men number of leves per plnt in control nd tretment plots in April of (Pilli Trce = , P = 0.51). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different Control Tretment Experimentl Tretment Figure 17. Men eril dimeter of plnts in control nd tretment plots in April of (Pilli Trce = , P = 0.51). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different.

33 Men Height (cm) Control Tretment Experimentl Tretment Figure 18. Men height of plnts in control nd tretment plots in April of (Pilli Trce = , P = 0.51). Error brs represent percent ± 1 SE. Letters bove brs re n indictor of significnce. Brs with the sme letters re not significntly different, nd brs with different letters re significntly different. Although there were no sttisticlly significnt differences in growth between control nd tretment plots in the second yer of growth, plnts in tretment plots were still lrger thn those in control plots. Men eril dimeter nd height of plnts were generlly greter for plnts in tretment plots, but these differences were not cptured by sttisticl nlysis. This ws most likely due to low numbers of surviving plnts in the second yer. There were only 14 surviving tretment plnts nd 13 surviving control plnts in The low smple sizes for this yer incresed the possibility of Type I error beyond threshold where differences could be found significnt. To summrize, results showed tht tretment with AMF inocultion hs positive effect on growth of Aboroni mcrocrp following germintion. Since there were lso

34 24 significnt effects on plnt growth due to trnsect nd plot, results indicte tht A. mcrocrp is extremely sensitive to vritions in microhbitt. Inocultion hd no significnt effect on germintion or survivorship nd no effect on developmentl stge in either yer. Although there ws greter growth shown from plnts in tretment plots in the second yer, these differences were not significnt. Exmintion of Nturlly Occurring Mycorrhize Speedbll Super Blck Indi Ink (wterproof), Bomby Blck Ink (wterproof), nd Higgins Blck Ink (wterproof) ech immeditely precipitted out of solution once vinegr ws dded. I therefore determined tht none of these inks were suitble for use in this stining procedure. Higgins Blck Ink (non-wterproof) nd Sheffer Blck ink (non-wterproof) both styed soluble in the vinegr solution nd were suitble for stining. Root segments stined in the Higgins (non-wterproof) solution hd moderte contrst nd ppered drk nd slightly blurry under mgnifiction (Fig. 19). Fungl structures were stined drk blck, while the surrounding root tissue ws comprtively more trnslucent, but difficult to see through. This stin combintion ppered to be highly soluble nd kept bleeding out of root smples, drkening the wter even fter being stored for weeks nd rinsed gin. This mde working with the root smples slightly more difficult, but hd no effect on my bility to detect fungl structures under microscopic observtion. The degree of contrst with Sheffer ink (non-wterproof) ws excellent (Fig. 19b). Roots stined in this solution remined cler while fungl structures were stined

35 25 reddish brown or occsionlly bright blue. This result differs from those obtined by Vierheilig et l. (1998) in which AMF structures ppered blck fter stining with Sheffer blck ink (non-wterproof). b Figure 19. Lentil root smples with observble AMF coloniztion. Microgrphs tken t 10x mgnifiction fter stining with either () Higgins blck ink (non-wterproof) or (b) Sheffer blck ink (non-wterproof). Arrows indicte fungl structures within the root tissue. Infection in smples stined with Higgins blck ink (non-wterproof) ws 90% (± 3.84%) nd infection in smples stined with Sheffer blck ink (non-wterproof) ws 96% (± 5.88%). This indictes no difference in the utility of these ink brnds for the purposes of detecting AMF infections. However, the clrity nd degree of contrst were better with Sheffer brnd ink (non-wterproof). Since Sheffer ink (non-wterproof) provides better stining, it ws used for stining the root smples collected from the experimentl site.

36 26 AMF infection rtes were 66.3% (± 6.94%) in Plntego sp., 60.1% (± 7.19%) in Rhododon cilitus, nd 46.5% (± 11.6%) in Croton rgyrnthemus (Figure 20). This indictes tht there is reltively strong preexisting mycorrhizl component to the community structure t the experimentl field site. b c Figure 20. Nturlly occurring mycorrhize fter stining with Sheffer blck ink (nonwterproof). () Croton rgyrnthemus roots t 40x mgnifiction. Drk brown res indicte fungl structures. (b) Plntego sp. roots t 40x mgnifiction. Bright blue res indicte fungl structures. (c) Rhododon cilitus roots t 40x mgnifiction. Drk brown res indicte fungl structures.

37 CHAPTER IV DISCUSSION The importnce of rbusculr mycorrhizl fungi for improving the fitness of vsculr plnts hs been well-known for decdes (Allen, 1991; Smith nd Red, 1997). Endngered nd thretened plnt species my be ble to benefit from this ssocition if AMF inocultions re utilized in their recovery plns. A study of multiple endngered Hwiin plnts reveled tht individuls inoculted with AMF were generlly lrger nd hd higher survivorship thn control plnts (Gemm nd Koske, 1995). A seprte study of four more endemic Hwiin plnts, two of which re endngered, yielded similr results, showing tht inoculted plnts hd higher P levels in their tissues, hd higher root biomss, nd hd shoots tht were up to seven times lrger thn plnts tht were not inoculted (Gemm et l., 2001). In nother study, two endngered plnt species locted in south Florid, Jcquemonti reclint nd Amorph crenult, were both reported to hve crucil reltionships with AMF (Fisher nd Jychndrn, 2002). These species displyed fitness benefits such s incresed seedling growth nd greter P uptke when inoculted with mycorrhizl fungi (Fisher nd Jychndrn, 2002). The endngered plnts Pulstill slvic nd Plntgo trt, s well s the ecologiclly extinct Senecio umbrosus ll hd positive responses to AMF inocultion (Zubek et l., 2009). In this study the inoculted specimens displyed incresed efficiency for energy conservtion 27

38 28 nd incresed stbility s well s enhnced growth, photosynthetic ctivity, nd nutrient content (Zubek et l., 2009, p. 121). In more extreme cses, species cn be completely dependent on mycorrhizl ssocitions nd cnnot survive without them. Astrglis pplegtei, nother endngered species, is ble to survive in rtificil or sterilized substrtes only when inoculted with soil contining AMF (Brroetven et l., 1998). Cses such s this illustrte how criticl it cn be to hve knowledge on the mycorrhizl sttus of plnt species of concern when developing conservtion strtegies. In ddition to incresing nutrient uptke in deficient soils, mycorrhize hve the bility to reduce the thret of toxic ions in sline nd lkline soils (Zhng et l., 2011). Specificlly, the incresed P uptke provided by AMF coloniztion cn mitigte the negtive effects of slinity by equlizing concentrtions of N nd Cl ions (Zhng et l., 2011). There ws n observble effect on growth nd germintion of A. mcrocrp in However, significnt differences in percent germintion were due to trnsect, not inocultion. This could hve been due to subtle differences in moisture levels, soil nutrient vilbility, preexisting microbiot ssemblges, or other uncontrolled environmentl fctors. Although I ttempted to estblish three trnsects tht were s uniform s possible by plcing them within severl meters of one nother nd by plcing them prllel to one nother, the subtle differences tht existed between them were enough to produce differences in germintion success. There were no observble differences in survivorship from Mrch 2011 to Mrch 2012 due to tretment or trnsect. Growth ws improved due to inocultion. Lef counts on inoculted plnts were higher

39 29 thn control plnts in Mrch nd April of 2011, suggesting n dvntge in photosynthetic potentil for these individuls. This result is in greement with other studies such s Zubeck et l. (2009) in which inoculted plnts showed incresed efficiency for photosynthesis. Aeril dimeters of inoculted plnts in April of 2011 were lso greter thn control plnts, once gin suggesting incresed photosynthetic cpbility due to higher surfce re. These dvntges in size nd photosynthetic cpbilities in the first growth seson could provide more energy for plnts to estblish robust tproot systems erly in life. The three counties where A. mcrocrp is known to occur hve n verge nnul precipittion between 96.5 nd 99.8 centimeters (Nturl Fibers Informtion Center 1987), but the pst few yers hve been unusully dry. Throughout 2011, Freestone County received only centimeters of precipittion (Ntionl Climtic Dt Center, 2012). During Mrch of 2011, vrious prts of Freestone County were simultneously t drought intensities of D1 (moderte), D2 (severe), nd D3 (extreme), progressing to D3 for the entire county by the end of April (Ntionl Climtic Dt Center, 2012b). Leon nd Robertson counties were lredy in D3 t the beginning of Mrch 2011 nd hd progressed to D4 (exceptionl) by the end of April 2011 (Ntionl Climtic Dt Center, 2012b). Since both plnts nd fungi re highly moisturedependent, the lck of rinfll my hve impcted growth nd survivorship s well s prevented thorough AMF coloniztion from the inoculnt. In fct, there is evidence tht mycorrhizl fungi hve the potentil to become prsitic to their host plnts when environmentl conditions re stressful nd the cost of symbiosis becomes too gret (Johnson et l., 1997; Krst et l., 2008). Hd the mount of precipittion t the

40 30 experimentl site more closely reflected norml ptterns throughout the course of this experiment, there my hve been more of n effect due to AMF inocultion, nd the observed effects my hve been significnt in the second yer of growth. Furthermore, there were lredy mycorrhizl fungi present in the hbitt preceding the experiment, so the ddition of more my hve hd little to no effect. In the second yer of growth (2012), there were no longer ny sttisticlly significnt benefits in growth due to inocultion. If there hd been higher survivorship in this study, differences between growth of plnts in tretment nd control plots might hve been detected by the sttisticl nlyses. Since smple sizes were so low in the second yer, it would hve been very difficult to find significnt results. No difference in survivorship ws observed between control nd tretment plots either. Once gin, this my hve been due to unfvorble environmentl conditions. However, this phenomenon is evidently not unherd of in res where mycorrhize re lredy present. A study evluting the effectiveness of AMF inocultions for the restortion of rodside priries found tht lthough there were coloniztion benefits in the first growth seson, there ws no difference between inoculted nd control plots fter 27 months (White et l., 2008). Inoculnt from the tretment plots, or the preexisting mycorrhize in the re, could hve spred to control plots by tht time. In other words, the inoculnt my hve served s jump-strt for coloniztion, but the nturlly occurring mycorrhize in the re would hve colonized control plnts eventully. Inocultion my hve only sped up the process, which my hve been the cse in this study s well.

41 31 Observble benefits provided by mycorrhizl symbiosis vry gretly bsed on fungl species, plnt species, nd hbitt types (Smith nd Red, 1997). They cn even vry mong members of the sme genus (Sigueir nd Sggin-Junior, 2001). Generliztions concerning AMF dependency should therefore be voided, nd ech species of concern should be evluted individully. Lrge-scle inocultions cn be time consuming nd costly, so some uthors dvise tht mycorrhizl inocultions be utilized only when existing AMF coloniztion is lcking or when soil nutrient levels re low (White et l., 2008). This study hs shown tht AMF inocultion hs the potentil to improve growth in A. mcrocrp, especilly in the first yer, suggesting tht mycorrhizl inocultion could be desirble prt of reintroduction plnt for this species in the future. Further evlution is necessry to determine the utility of inocultions in the context of reintroduction procedures. In the coming yers, I would recommend reserch tht investigtes the effectiveness of different inocultion methods with A. mcrocrp, such s trenching, drilling, brodcsting, or even the use of different mycorrhizl species. I would lso recommend reserch to explore how long there is n observble benefit to mycorrhizl inocultion for vrious plnt species in field conditions. Finlly, I would like to see studies tht determine if A. mcrocrp is ecologiclly dependent upon mycorrhize, or if they re negtively ffected by being plnted in soils tht hve been treted with fungicide or sterilized.

42 LITERATURE CITED Allen, M.F The Ecology of Mycorrhize. Sn Diego Stte University. Cmbridge University Press. New York, NY. Brroetven, C., S.D. Gisler, D.L. Luom, nd R.J. Meinke Mycorrhizl sttus of the endngered species Astrglus pplegtei Peck s determined from soil biossy. Mycorrhiz. 8: Flk, D.A. nd P. Olwell Scientific nd policy considertions in restortion nd reintroduction of endngered species. Rhodor. 94: Fisher, J.B. nd K. Jychndrn Arbusculr mycorrhizl fungi enhnce seedling growth in two endngered plnt species from South Florid. Interntionl Journl of Plnt Sciences. 163: Gllowy, L Abroni mcrocrp (Nyctgincee): new species from Texs. Brittoni. 24: Gemm, J.N. nd R.E. Koske Vesiculr-rbusculr mycorrhizl inocultion of Hwiin plnts: conservtion technique for endngered tropicl species. Pcific Sci. 49: Gemm, J.N., R.E. Koske, nd M. Hbte Mycorrhizl dependency of some endemic nd endngered Hwiin plnt species. Americn Journl of Botny. 89: Goodson, J.J. nd P.S. Willimson Germintion of seeds in the endngered Abroni mcrocrp. Southwestern Nturlist. 56: Hrley, J.L. nd S.E. Smith Mycorrhizl Symbiosis. Acdemic Press. New York, NY. Johnson, N.C., J.H. Grhm, nd F.A. Smith Functioning of mycorrhizl ssocitions long the mutulism-prsitism continuum. New Phytologist. 135: Krst, J., L. Mrczk, M.D. Jones, nd R. Turkington The mutulism-prsitism continuum in ectomycorrhizs: quntittive ssessment using met-nlysis. Ecology. 89:

43 33 Meredith, C Recovery of Abroni mcrocrp following popultion disturbnce. Msters Thesis. Texs Stte University-Sn Mrcos. Ntionl Climtic Dt Center Monthly Climtologicl Summry: Sttion: GHCND:US1TXFT0001, Okwood 4.2 NE, TX US. Ntionl Climtic Dt Center. 2012b. NIDIS Mp nd Dt Viewer. Retrieved June2012 vi Nturl Fibers Informtion Center The Climtes of Texs Counties. University of Texs Bureu of Business Reserch, Grdute School of Business, Austin, TX. Novcek, M.J. nd E.E. Clelnd The current biodiversity extinction event: Scenrios for mitigtion nd recovery. Proceedings of the Ntionl Acdemy of Sciences of the United Sttes of Americ. 98: Pnwr, J. nd A. Vys AM fungi: A biologicl pproch towrds conservtion of endngered plnts in Thr desert, Indi. Current Science. 82: Pvlik, B.M Defining nd mesuring success. In Restoring Diversity: Strtegies for Reintroduction of Endngered Plnts. (eds. Flk, D.A., C.I. Millr, nd M. Olwell), pp Islnd Press, Wshington, DC. Plenchette, C., J.A. Fortin, nd V. Furln Growth responses of severl plnt species to mycorrhize in soil of moderte P-fertility. Plnt nd Soil. 70: R Development Core Tem R: A lnguge nd environment for sttisticl computing. R Foundtion for Sttisticl Computing, Vienn, Austrli. ISBN , URL Sebloom, E.W., A.P. Dobson, nd D.M. Stoms Extinction rtes under nonrndom ptterns of hbitt loss. Proceedings of the Ntionl Acdemy of Science of the United Sttes of Americ. 99: Sigueir, J.O. nd O.J. Sggin-Junior Dependency on rbusculr mycorrhizl fungi nd responsiveness of some Brzilin ntive woody species. Mycorrhiz. 11: Smith, S.E. nd D.J. Red Mycorrhizl symbiosis. 2nd ed. Sn Diego, Cliforni: Acdemic Press. 605 p. U.S. Fish nd Wildlife Service Endngered nd thretened wildlife nd plnts: determintion of endngered sttus for Abroni mcrocrp (lrge-fruited sndverben). Federl Register. 43:

44 34 U.S. Fish nd Wildlife Service Lrge-fruited snd verben (Abroni mcrocrp) recovery pln. Albuquerque, New Mexico. Vierheilig, H., A.P. Coughln, U. Wyss, nd Y. Piche Ink nd Vinegr, Simple Stining Technique for Arbusculr-Mycorrhizl Fungi. Applied nd Environmentl Microbiology. 64: White, J.A., J. Tllksen, nd I. Chrvt The effects of rbusculr mycorrhizl fungl inocultion t rodside pririe restortion site. Mycologi. 100: Willimson, P.S Section 6 Grnt Finl Report: Protection on Privte Lnds nd Reserch for Recovery of Lrge-fruited Snd-verben. (USFWS Coopertive Agreement No ). 48pp. Willimson, P.S. nd C.R. Werth Levels nd ptterns of genetic vrition in the endngered species Abroni mcrocrp (Nyctgincee). Americn Journl of Botny. 86: Willimson, P.S., nd S.K. Bzeer Self-incomptibility in Abroni mcrocrp (Nyctgincee). The Southwestern Nturlist. 42: 4: Willimson, P.S., L. Mulini, nd G. Jnssen Pollintion biology of Abroni mcrocrp (Nyctgincee), n endngered Texs species. Southwestern Nturlist. 39: Zhng, Y.F., P. Wng, Y.F. Yng, Q. Bi, S.Y. Tin, nd X.W. Shi Arbusculr mycorrhizl fungi improve reestblishment of Leymus chinensis in bre slinelkline soil: Impliction on vegettion restortion of extremely degrded lnd. Journl of Arid Environments. 75: Zndvlli, R.B., L.R. Dillenburg, nd P.V.D. De Souz Growth responses of Arucri ngustifoli (Arucricee) to inocultion with the mycorrhizl fungus Glomus clrum. Applied Soil Ecology. 25: Zubek, S., K. Turnu, M. Tsimilli-Michel, nd R.J. Strsser, Response of endngered plnt species to inocultion with rbusculr mycorrhizl fungi nd soil bcteri. Mycorrhiz. 19: