Colletotrichum truncatum NRRL 13737

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p /90/ $02.00/0 Copyright C) 1990, Americn Society for Microbiology Vol. 56, No. 11 Crbon Concentrtion nd Crbon-to-Nitrogen Rtio Influence Submerged-Culture Conidition by the Potentil Bioherbicide Colletotrichum trunctum NRRL MARK A. JACKSON* AND RODNEY J. BOTHAST Fermenttion Biochemistry Reserch, Northern Regionl Reserch Center, Agriculturl Reserch Service, U.S. Deprtment of Agriculture, 1815 North University Street, Peori, Illinois Received 25 June 1990/Accepted 31 August 1990 We ssessed the influence of vrious crbon concentrtions nd crbon-to-nitrogen (C:N) rtios on Colletotrichum trunctum NRRL conidium formtion in submerged cultures grown in bsl slts medium contining vrious mounts of glucose nd Csmino Acids. Under the nutritionl conditions tested, the highest conidium concentrtions were produced in medi with crbon concentrtions of 4.0 to 15.3 g/liter. High crbon concentrtions (20.4 to 40.8 g/liter) inhibited sporultion nd enhnced the formtion of microsclerotiumlike hyphl msses. At ll the crbon concentrtions tested, culture grown in medium with C:N rtio of 15:1 produced more conidi thn cultures grown in medi with C:N rtios of 40:1 or 5:1. While glucose exhustion ws often coincident with conidium formtion, cultures contining residul glucose sporulted nd those with high crbon concentrtions (>25 g/liter) exhusted glucose without sporultion. Nitrogen source studies showed tht the levels of C. trunctum NRRL conidition were similr for ll protein hydrolystes tested. Reduced conidition occurred when mino cid nd inorgnic nitrogen sources were used. Of the nine crbon sources evluted, cette s the sole crbon source resulted in the lowest level of sporultion. The use of fungl plnt pthogens to control weeds which re not sfely or economiclly mnged by chemicl herbicides is becoming prcticble option (7). Two commercil products re Colletotrichum gloeosporioides f. sp. eschynomene spores (Collego; Ecogen, Inc.) for the control of northern jointvetch (Aeschynomene virginic) in Arknss rice fields nd Phytophthor plmivor (Devine; Abbott Lbortories) for the control of strnglervine (Morreni odort) in Florid citrus groves (2, 6). Numerous Colletotrichum species show promise for use s mycoherbicides. Strins of Colletotrichum mlvrnum nd Colletotrichum coccodes which show promise s biocontrol gents ginst prickly sid (Sid spinos) nd velvetlef (Abutilon theophrsti), respectively, hve been identified (4, 9). Strins of C. gloeosporioides which specificlly infect the noxious weeds Clidemi hirt nd Mlv pussill hve been discovered (5, 8). Hemp sesbni (Sesbni exltt) is troublesome weed in the southern United Sttes nd cuses considerble losses in production of cotton, soybens, nd rice. Boyette (1) recently discovered strin of Colletotrichum trunctum, NRRL 13737, which specificlly infects nd kills Sesbni exltt but which is not menble to submerged-culture sporultion with trditionl liquid culture medi. The bility to economiclly produce fungl spores is requirement for the commercil development of bioherbicide. Our reserch is directed towrd the development of costeffective methods for producing fungl spores. In order to optimize submerged-culture sporultion of C. trunctum, we evluted the effects of vrious nutritionl environments on conidium formtion. In this pper we report the development of semidefined medium for the growth nd sporultion of C. trunctum NRRL in submerged culture nd * Corresponding uthor the influence of crbon concentrtion nd crbon-to-nitrogen (C:N) rtio on sporultion. MATERIALS AND METHODS Orgnism. C. trunctum (Schw.) Andrus nd Moore (NRRL [deposited by D. Boyette, USDA, Stoneville, Miss.]) ws obtined from the U.S. Deprtment of Agriculture/Agriculturl Reserch Service Northern Regionl Reserch Center Culture Collection. Stock cultures of C. trunctum NRRL were grown s single-spore isoltes on potto dextrose gr t room temperture nd stored s 1-mm potto dextrose gr plugs in 10% glycerol t -80 C. For liquid-culture studies, spore inocul were produced by growing glycerol stock cultures of C. trunctum on potto dextrose gr pltes t room temperture. Spore inocul were obtined by rinsing the sporulted potto dextrose gr pltes with deionized wter. Medi. The defined bsl slts medium ws composed of KH2PO4, 2.0 g/liter; MgSO4 7H20, 0.3 g/liter; CCl2.2H20, 0.4 g/liter; ZnSO4-7H20, 14 mg/liter; MnSO4- H20, 16 mg/ liter; FeSO4-7H20, 50 mg/liter; CoCI2 6H20, 37 mg/liter; thimine, riboflvin, pntothente, nicin, pyridoxmine, nd thioctic cid, 500,ug of ech per liter; nd folic cid, biotin, nd vitmin B12, 50 pug of ech per liter in deionized wter. Stock solutions of glucose (20%, wt/vol; Difco Lbortories, Detroit, Mich.) nd vitmin-free Csmino Acids (5%, wt/vol; Difco) were dded to the bsl slts medium s required. Glucose nd other crbon source stock solutions were utoclved seprtely. A ph of 5.5 ws mintined by the ddition of either 2 N NOH or 2 N HCI. Crbon concentrtion nd C:N rtio studies were conducted with the bsl slts medium supplemented with vrious mounts of glucose nd Csmino Acids s the crbon nd nitrogen sources. Crbon concentrtions nd C:N rtios were clculted s the crbon nd nitrogen present in the glucose (40% crbon) nd Csmino Acids Downloded from on August 31, 2018 by guest

2 3436 JACKSON AND BOTHAST (53% crbon, 16% nitrogen). During C:N rtio studies, crbon concentrtions were mintined t constnt level by vrying the mount of glucose nd Csmino Acids present. For nitrogen source studies, our bsl slts medium ws supplemented with glucose nd vrious nitrogen sources t crbon concentrtion of 8 g/liter nd C:N rtio of 15:1. Nitrogen sources tested were vitmin-free Csmino Acids, mlt extrct, yest extrct (Difco), cottonseed hydrolyste, distillers grins nd solubles, bovine serum lbumin, corn steep liquor, corn gluten hydrolyste, sprgine, glutmte (Sigm Chemicl Co., St. Louis, Mo.), digest of milk nd met protein (AY19BT), Enhncetone (PP9OBT), ppic digest of soy (SE50BT), digest of csein (CE9OBT), digest of rice flour (RE30BT), digest of niml tissue (AE80BT; Deltown Chemurgic, Greenwich, Conn.), sodium nitrte, ure, nd mmonium sulfte (Fisher Scientific, Firlwn, N.J.). A bsl slts medium ws supplemented with Csmino Acids nd vrious crbon sources t crbon concentrtion of 8 g/liter nd C:N rtio of 15:1 in our crbon source studies. Crbon sources tested were glucose nd sucrose (Difco); fructose, glycerol, nd cette (Fisher); glctose, cellulose, nd corn strch (Sigm); nd citrte (J. T. Bker Chemicl Co., Phillipsburg, N.J.). Modified Richrd V-8 medium consisted of sucrose, 50 g/liter; KNO3, 10 g/liter; KH2PO4, 5 g/liter; MgSO4 7H20, 2.5 g/liter; FeCl2, 0.02 g/liter; nd mixed vegetble juice, 150 ml/liter in deionized wter, s previously described (2). The medium ws djusted to ph of 6.0 with 5 N NOH prior to utoclving. Potto dextrose broth (Difco) ws prepred ccording to the instructions of the mnufcturer, with deionized wter. Submerged culture. Liquid-culture experiments were crried out in duplicte 250-ml Erlenmeyer flsks (no ; Bellco Glss, Inc., Vinelnd, N.J.) t 100-ml volume. The cultures were incubted t 28 C nd 300 rpm in rotry shker incubtor. The initil spore concentrtion for ll submerged cultures ws 5 x 104 spores per ml. The ph of ech medium ws mintined by dily djustments with either 2 N NOH or 2 N HCl. Flsks were shken frequently to inhibit mycelil growth on the flsk wll. Anlyses. Glucose concentrtions were determined by using high-performnce liquid chromtogrphy system (Spectr-Physics, Sn Jose, Clif.) equipped with n Aminex ion exclusion column (model HXP-87H; Bio-Rd Lbortories, Richmond, Clif.) nd refrctive index detector (model 410; Wters Assocites, Inc., Milford, Mss.). The column ws operted t room temperture, nd cidified wter ( N H2SO4) ws used s the mobile phse. Spore concentrtions were determined microscopiclly on n Olympus BH-Z microscope using Neubuer hemocytometer (Reichrt-Jung, Bufflo, N.Y.). Homogeneous smples for counting spores were obtined by vortexing the smple for 30 s before the hemocytometer ws loded. RESULTS Nitrogen nd crbon source studies. Nineteen nitrogen sources were evluted for their influence on conidium formtion in submerged cultures of C. trunctum NRRL All the nitrogen sources tested supported vigorous growth, with the exception of mmonium sulfte. Cultures supplemented with mmonium sulfte grew slowly nd did not sporulte. The 14 cultures supplemented with complex nitrogen sources, those contining protein nd protein hy- APPL. ENVIRON. MICROBIOL. TABLE 1. Submerged-culture conidition of C. trunctum NRRL with vrious nitrogen sources Men no. of Men residul Nitrogen source conidi/ml glucose (mg/ml) Csmino Acids 3.9 x Digest of milk & met protein 3.3 x 107 b 2.3 Cottonseed hydrolyste 3.3 x 107 b 0.7 Enhncetone 2.7 x 107 b 0.8 Ppic digest of soy 2.6 x 107 b 0.6 Distillers' grins nd solubles 1.8 x 107 bc 0.8 Digest of csein 2.2 x 107 bc 0.8 Mlt extrct 2.5 x 107 bc 17.2 Bovine serum lbumin 1.6 x 107 bc ND" Yest extrct 1.2 x 107 bc 0.9 Digest of rice flour 1.0 x 107 bc 3.3 Corn steep liquor 9.0 x 106 bc ND Digest of niml tissue 1.4 x 107 bc 0.8 Corn gluten hydrolyste 6.5 x 106 bc 1.1 Sodium nitrte 6.0 x 106 bc ND Asprgine 3.5 x 106 c ND Glutmte 2.7 x 106 c ND Ure 1.0 x 106 d ND Ammonium sulfte d " Six-dy-old cultures with crbon concentrtion of 8 g/liter nd C:N rtio of 15:1. b Vlues followed by different letters re significntly different (P < 0.05) using Fisher's protected lest significnt difference on the log1o of the vlues ' ND, Not done. d _, Poor growth (no vlue obtined). drolystes, produced sttisticlly similr conidium concentrtions (6.5 x 106 to 3.9 x 107 spores per ml) following 6 dys of growth (Tble 1). Although cultures supplemented with sodium nitrte, sprgine, nd glutmte produced significntly fewer spores thn cultures supplemented with Csmino Acids, spore levels were not significntly lower thn those of mny of the complex nitrogen-supplemented cultures (Tble 1). Cultures supplemented with ure nd mmonium sulfte produced significntly fewer conidi thn cultures supplemented with ll other nitrogen sources tested. Seven of the nine crbon sources tested gve similr levels of sporultion in submerged cultures of C. trunctum NRRL (Tble 2). Spore production in cette-supplemented cultures ws significntly reduced compred with spore production in cultures with the other crbon sources tested. TABLE 2. Submerged-culture conidition of C. trunctum NRRL in bsl slts medium supplemented with Csmino Acids nd vrious crbon sources Men of Crbon source conidi/ml' bno.mof Corn strch x 107 Fructose x 107 Sucrose x 107 Glycerol x 107 Glucose x 107 Cellulose x 106 b Citrte x 106 b Glctose x 104 b Acette x 103 c Six-dy-old culture with crbon concentrtion of 8 g/liter nd C:N rtio of 15:1. b Vlues followed by different letters re significntly different (P < 0.05) using Fisher's protected lest significnt difference on the log1o of the vlues Downloded from on August 31, 2018 by guest

3 VOL. 56, 1990 TABLE 3. Influence of crbon concentrtion on C. trunctum NRRL conidi production Initil Dy 5 Dy 6 Dy 7 Crbonglcs (g/liter) glucose Glucose Conidi Glucose Conidi Glucose Conidi (g/liter) (g/liter) (106/ml)b (g/liter) (106/ml)' (g/liter) (106/ml)b b b c b b d c c d c c d c c d c c C:N rtio, 15:1. b Vlues followed by different letters re significntly different (P < 0.05) using Fisher's protected lest significnt difference on the loglo of the vlues In generl, cultures supplemented with cette, glctose, citrte, nd cellulose produced low levels of conidi compred with those supplemented with glucose, sucrose, fructose, glycerol, nd corn strch. Crbon concentrtion nd C:N rtio studies. When medi with C:N rtio of 15:1 were used, conidition ws mesured in cultures with crbon concentrtions between 5.1 nd 40.8 g/liter (Tble 3). Conidition in 5-dy-old cultures ws highest, with crbon concentrtion of 10.2 g/liter, while rnge of crbon concentrtions (5.1 to 15.3 g/liter) gve the highest levels of conidition in 6- nd 7-dy-old cultures. Cultures with crbon concentrtions of 25.5 g/liter or more did not sporulte. Glucose ws bsent from the superntnt of ll cultures following 7 dys of growth, regrdless of the initil glucose concentrtion. Under the conditions of this study, incresed crbon concentrtion ws correlted to incresed hyphl melniztion (Fig. 1). Microscopic exmintion of these cultures showed tht hyphl melliztion occurred in microsclerotiumlike, compct hyphl msses. An increse in the number of these highly melnized hyphl msses ws correlted to reduced conidium formtion. The effect of C:N rtio on conidium formtion ws s- C. TRUNCATUM NRRL CONIDIATION 3437 TABLE 4. Influence of C:N rtio on conidition by C. trunctum NRRL t vrious crbon concentrtions C:N rtio Spores/ml (lo5) t crbon concn (g/liter) : :1 34 c 104 c 138 b 15:1 57 c 150 c 209 b 10:1 31 c 143 c 132 b 7.5:1 11 b 99 C 88 b 5:1 13 b 41 b 62 Vlues followed by different letters re significntly different (P < 0.05) using Fisher's protected lest significnt difference on the loglo of the vlues sessed by growing C. trunctum NRRL cultures t constnt crbon concentrtions (2, 4, nd 8 g/liter) with C:N rtios of 40:1, 20:1, 15:1, 10:1, 7.5:1, nd 5:1. A comprison of conidium production for 6-dy-old cultures grown in medi with the sme crbon concentrtion nd different C:N rtios showed tht medium with C:N rtio of 15:1 produced significntly more conidi thn medi with C:N rtios of 40:1 nd 5:1, regrdless of the crbon concentrtion (Tble 4). Conidium production in medi with C:N rtios from 20:1 to 10:1 ws not significntly different for cultures grown with 2 g of crbon per liter. In cultures with 4 or 8 g of crbon per liter, medi with C:N rtios between 20:1 nd 7.5:1 produced similr numbers of conidi (Tble 4). A comprison of conidium production for cultures grown with vrious crbon concentrtions nd the sme C:N rtio showed tht medi with 4 nd 8 g of crbon per liter produce similr concentrtions of conidi which re significntly higher thn those of cultures grown with 2 g of crbon per liter (Fig. 2). Only t C:N rtio of 40:1 did cultures grown with 2 nd 4 g of crbon per liter produce similr levels of conidi which were lower thn those produced by cultures with 8 g of crbon per liter. DISCUSSION The evlution of nutritionl fctors on sporultion by C. trunctum NRRL in submerged culture required the 30 E 20 Downloded from on August 31, 2018 by guest FIG. 1. Six-dy-old C. trunctum NRRL submerged cultures grown t C:N rtio of 15:1 with vrious glucose concentrtions. Incresing glucose concentrtions inhibit conidition (Tble 3) nd increse the production of melnized, microsclerotiumlike hyphl msses. 10 b b: b ~~~~~~~~~~ 40:1 20:1 15:1 10:1 7.5:1 5:1 C/N Rtio FIG. 2. The influence of crbon concentrtion on conidition by 6-dy-old C. trunctum NRRL submerged cultures grown t vrious C:N rtios. Within ech C:N rtio grouping, men spore concentrtions with different letters re significntly different (P < 0.05) using Fisher's protected lest significnt difference on the loglo of the vlues Symbols (grms of crbon per liter): El, 2;, 4; E,8.

4 3438 JACKSON AND BOTHAST development of defined medium which could support good growth nd sporultion. Using vitmin-enriched bsl slts medium which hd been shown to support good growth by other fungi (3), we evluted the suitbility of vrious crbon nd nitrogen sources (Tbles 1 nd 2). Results from these experiments showed tht nitrogen sources contining protein or protein hydrolystes supported similrly high levels of conidium production, while inorgnic nitrogen sources produced lower levels of conidition. Vitmin-free Csmino Acids were used in ll other experiments becuse of their vitmin-free nture nd their reltive consistency in composition compred with other proteinceous nitrogen sources. Glucose ws used s the crbon source becuse of its bility to support good conidium formtion nd becuse of its ese of mesurement. Under the conditions of these experiments, crbon concentrtion nd C:N rtio were shown to influence submerged-culture conidium production by C. trunctum NRRL Optimum sporultion occurred when cultures were supplied with Csmino Acids nd glucose with totl crbon concentrtion of 4.0 to 15.3 g/liter t C:N rtio between 20:1 nd 7.5:1. A C:N rtio of 15:1 consistently produced the highest levels of conidition. The inhibition of conidium formtion by high crbon concentrtions (Tble 3) ws unexpected. The possibility tht residul glucose ws inhibiting sporultion ws dismissed when glucose ws found to be bsent from the culture superntnts following 7 dys of growth. Nitrogen source studies lso indicted tht glucose depletion ws unnecessry for sporultion (Tble 1). The production of numerous microsclerotiumlike, highly melnized, compct hyphl msses rther thn conidi in C. trunctum NRRL cultures grown t high crbon concentrtions suggests tht crbon concentrtion my ply n importnt developmentl role. As comprison, we tested conidium production by C. gloeosporioides f. sp. eschynomene t vrious crbon concentrtions. Cultures of C. gloeosporioides f. sp. eschynomene produced more conidi s crbon concentrtions were incresed beyond those levels which inhibited C. trunctum sporultion, without the production of microsclerotiumlike hyphl msses (dt not shown). These results suggest tht regultion of differentition by crbon concentrtion is not universl in Colletotrichum species. Richrd V-8 medium is typiclly used to grow nd produce spores of vrious Colletotrichum species in submerged culture. Boyette found tht C. trunctum NRRL sporulted poorly in Richrd V-8 medium nd produced numerous microscleroti (1). Under our culture conditions, C. trunctum NRRL produced significntly fewer spores (3 x 106 spores per ml) in Richrd V-8 medium thn in our defined medium (2 x 107 spores per ml). Anlysis of Richrd V-8 medium shows tht it supplies pproximtely 23 g of APPL. ENVIRON. MICROBIOL. crbon per liter, concentrtion which inhibited conidium formtion nd enhnced microsclerotium development by C. trunctum NRRL in our defined-medium studies. Conversely, potto dextrose broth (Difco) provides pproximtely 10 g of crbon per liter nd supports submergedculture sporultion by C. trunctum t levels comprble to those obtined with our defined medi. The influence of crbon concentrtion on the submerged-culture sporultion of C. trunctum NRRL in these complex medi followed the pttern we expected from our defined-medium studies. An understnding of how the nutritionl environment influences sporultion is required in order to predict wht types of low-cost complex medi my be suitble for spore production. Our results with semidefined medium show tht crbon concentrtion nd C:N rtio re importnt nutritionl fctors regulting the submerged-culture sporultion of C. trunctum NRRL Recent studies indicte tht these nutritionl fctors influence C. trunctum conidil vibility nd conidil efficcy in inciting disese in Sesbni exltt (D. A. Schisler, M. A. Jckson, nd R. J. Bothst, submitted for publiction). The commercil use of fungl spores s bioherbicides will require cost-effective production techniques which mximize both conidil numbers nd conidil efficcy. LITERATURE CITED 1. Boyette, C. D Efficcy nd host rnge of recently discovered fungl pthogen for biocontrol of hemp sesbni. Proc. South. Weed Sci. Soc. 41: Dniel, J. T., G. E. Templeton, R. J. Smith, Jr., nd W. T. Fox Biologicl control of northern jointvetch in rice with n endemic fungl disese. Weed Sci. 21: Jckson, M. A., P. J. Slininger, nd R. J. Bothst Effects of zinc, iron, coblt, nd mngnese on Fusrium moniliforme NRRL growth nd fusrin C biosynthesis in submerged cultures. Appl. Environ. Microbiol. 55: Kirkptrick, T. L., G. E. Templeton, D. 0. TeBeest, nd R. J. Smith, Jr Potentil of Colletotrichum mlvrnum for biologicl control of prickly sid. Plnt Dis. 66: Mortensen, K The potentil of n endemic fungus, Colletotrichum gloeosporioides, for biologicl control of round-leved mllow (Mlv pussill) nd velvetlef (Abutilon theophrsti). Weed Sci. 36: Riddings, W. H Biologicl control of strnglervine in citrus- resercher's view. Weed Sci. 34: Templeton, G. E Sttus of weed control with plnt pthogens, p In R. Chrudttn nd H. L. Wlker (ed.), Biologicl control of weeds with plnt pthogens. John Wiley & Sons, Inc., New York. 8. Trujillo, E. E., F. M. Ltterell, nd A. E. Rossi Colletotrichum gloeosporioides, possible biologicl control gent for Clidemi hirt in Hwiin forests. Plnt Dis. 70: Wymore, L. A., C. Poirier, A. K. Wtson, nd A. R. Gotlieb Colletotrichum coccodes, potentil bioherbicide for control of velvetlef (Abutilon theophrsti). Plnt Dis. 72: Downloded from on August 31, 2018 by guest

5 AUTHOR'S CORRECTION Crbon Concentrtion nd Crbon-to-Nitrogen Rtio Influence Submerged-Culture Conidition by the Potentil Bioherbicide Colletotrichurm trunctum NRRL MARK A. JACKSON AND RODNEY J. BOTHAST Fermenttion Biochemistry Reserch, Northern Regionl Reserch Center, Agriculturl Reserch Service, U.S. Deprtment of Agriculture, 1815 North University Street, Peori, Illinois Volume 56, no. 11, p : The crbon-to-nitrogen (C:N) rtios for medi formulted with Csmino Acids were clculted on the bsis of 16% nitrogen content in the Csmino Acids. The vitmin-free Csmino Acids (Difco) used in these experiments contined only 8% nitrogen. Therefore, the ctul C:N rtios for medi formulted with Csmino Acids re twice the vlues reported in this pper. 2781