Gibberellins and carotenoids in the wild type and mutants of Gibberella fujikuroi

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, No . 1991, p. 3378-3382 Vol. 57, No. 11 0099-2240/91/113378-05$02.00/0 Copy igh C 1991, Ame ican Socie y o Mic obiology Gibbe ellins and Ca o enoids in he Wild Type and Mu an s o Gibbe ella ujiku oi REYES CANDAU, JAVIER AVALOS, AND ENRIQUE CERDA OLMEDO* Depa amen o de Gene ica y Bio ecnia, Uni e sidad de Se illa, Se ille, Spain Recei ed 19 Feb ua y 1991/Accep ed 16 Augus 1991 A new sc eening p ocedu e was used o isola e 14 gib mu an s o Gibbe ella ujiku oi wi h modi ica ions in he p oduc ion o gibbe ellins. The p oduc ion o ca o enoids and gibbe ellins was in es iga ed in he gib mu an s and in ep esen a i e ca mu an s wi h a ious modi ica ions o ca o enoid biosyn hesis. The de e mina ions o gibbe ellins we e ca ied ou wi h a simpli ied luo escence me hod. One o he mu an s lacked bo h gibbe ellins and ca o enoids. In many mu an s he wo pa hways compensa ed each o he : an inc ease in he p oduc ion o one g oup o compounds was accompanied by a dec ease in he p oduc ion o he o he . Unde ce ain condi ions he compensa ion was quan i a i e when he ou pu o he wo pa hways was measu ed in moles o he common p ecu so , ge anylge anyl py ophospha e. a-Picoline, an inhibi o o lycopene cyclase in G. ujiku oi, inhibi s gibbe ellin biosyn hesis. O he agen s ha a ec he accumula ion o ca o enoids ha e no no iceable e ec on he accumula ion o gibbe ellins; such is he case wi h diphenylamine and ,-ionone, wo inhibi o s o phy oene dehyd ogena ion, and isible ligh , which s imula es ca o enogenesis. The gibbe ellins a e a g oup o na u al plan ho mones wi h a ious e ec s on g ow h and mo phogenesis (13) and p ac ical applica ions in ag icul u e and b ewing. The gib- be ellins a e indus ially pu i ied om he cul u e media o some s ains o he ascomyce e Gibbe ella ujiku oi (6, 12), a ice pa hogen whose impe ec s age is known as Fusa ium monili o me. Te penoids a e a la ge amily o na u al compounds ha includes he gibbe ellins, he ca o enoids, which a e wide- sp ead pigmen s made by plan s and by many ungi and bac e ia, and he s e ols, which a e uni e sal componen s o cell memb anes. Gibbe ellins and ca o enoids a e made om a common 20-ca bon p ecu so , ge anylge anyl py ophos- pha e (11). The mycelia o G. ujiku oi con ain he o ange ca o enoid neu ospo axan hin and i s p ecu so s, such as o ulene, -y-ca o ene, and phy oene. The ca o enoid concen- a ion depends on he cul u e condi ions, pa icula ly he medium, illumina ion, and empe a u e (3, 4). The p oduc- ion o ca o enoids is inhibi ed by he addi ion o dipheny- lamine, a-picoline, o ,B-ionone (2). Many colo mu an s o G. ujiku oi show al e a ions o ca o enoid biosyn hesis (1). The deep-o ange mu an s ha e much mo e neu ospo axan hin han does he wild ype. Albino mu an s lack ca o enoids o accumula e he colo less phy oene. Red mu an s accumula e o ulene. Pale-o ange mu an s a e simila o he wild ype in he ligh and accumu- la e app eciable amoun s o ca o enoids in he da k (3). Indus ial esea che s ha e es ablished he condi ions o gibbe ellin p oduc ion and ha e looked o high-yielding s ains. Ve y li le is known abou he gene ics o G. u- jiku oi, and only a ew mu an s wi h speci ic al e a ions in gibbe ellin biosyn hesis ha e been desc ibed (5). We de eloped simpli ied me hods ha allowed us o isola e mu an s de ec i e in gibbe ellin p oduc ion. Analyses o hese mu an s, he wild ype, and mu an s wi h al e ed ca o enoid p oduc ion illus a e he ela ionships be ween he gibbe ellin and ca o enoid pa hways. * Co esponding au ho . MATERIALS AND METHODS S ains and gene al p ocedu es. The s ains used in his wo k a e lis ed in Tables 1 and 2. Unless o he wise s a ed, cul u es we e g own a 30°C in an o bi al shake (120 pm) in he da k in 500-ml E lenmeye lasks wi h 250 ml o liquid 10% ICI medium (10), a medium o gibbe ellin p oduc ion wi h 0.48 g o NH4NO3 pe li e . Whe e indica ed, he glass op o he incuba o allowed exposu e o he cul u e lasks o whi e ligh ( luence a e, 2 W m-2) om i e luo escen lamps (Syl ania F40T121D). o-Picoline was dissol ed in dis illed wa e and added o he medium a a inal concen- a ion o 2 g li e -1 ei he immedia ely be o e inocula ion o a e 9 days o g ow h. Diphenylamine was dissol ed in 96% e hanol and added o 9-day-old cul u es a inal concen- a ions o 20.3 mg o diphenylamine and 0.6 ml o e hanol pe li e . 3-Ionone was emulsi ied in a mix u e o e hanol and polyoxye hyleneso bi an monoolea e (Tween-80) and added o 9-day-old cul u es a inal concen a ions o 0.2 g o P-ionone, 2 ml o e hanol, and 1 ml o Tween-80 pe li e . The glucose concen a ions in he medium we e de e - mined wi h a glucose analyze (model 27; Yellow Sp ings Ins umen s, Yellow Sp ings, Ohio) o wi h glucose oxidase (9). Ca o enoid analyses we e ca ied ou as desc ibed p e i- ously (2). The e m "in e media e ca o enes" designa es he sum o colo ed ca o enes; i does no include neu ospo a- xan hin. Fo he compa ison o ca o enoid and gibbe ellin p oduc ion, i was assumed ha 1 mol o he p ecu so ge anylge aniol yields 267 g o phy oene, 265 g o in e me- dia e ca o enes, o 250 g o neu ospo axan hin (hal o a mole in each case) o 345 g o gibbe ellins (a mole o gibbe ellic acid). Fluo ime ic de e mina ion o gibbe ellins. Fluo escen de- i a i es o gibbe ellins we e ob ained by shaking oge he 0.2 ml o cul u e medium, 0.2 ml o e hanol (96%, ol/ ol), and 2 ml o a cooled mix u e o equal olumes o sul u ic acid and 96% e hanol and incuba ing he eac ion mix u e a 48°C o 30 min. Fluo escence spec a we e ob ained wi h an LS-5 Pe kin-Elme luo ime e . The luo escence emissions a 464 nm o samples exci ed a 406 nm we e compa ed wi h ha o au hen ic gibbe ellic acid (Sigma Chemical Co., S . 3378 GIBBERELLINS AND CAROTENOIDS IN GIBBERELLA MUTANTS 3379 TABLE 1. P oduc ion o gibbe ellins and ca o enoids in he wild- ype IM158289 and in gib mu an sa Gibbe ellins (mg Ca o enoids (jig g o d y w -') S ain 6 days 12 days Phy oene In e media e Neu ospo- ca o enes axan hin IM158289 22 79 1 6 3 SG121 3 10 0 11 7 SG122 7 27 2 19 12 SG123 1 15 09 3 SG124 2 25 0 5 3 SG127 5 15 0 0 0 SG128 6 32 0 8 4 SG129 7 23 0 3 2 SG133 15 49 0 2 1 SG134 10 57 0 6 1 SG135 4 5 1 18 6 SG136 4 82 22 15 SG137 00 0 6 1 SG138 00 2 19 16 SG139 0 1 2 14 9 a All cul u es we e incuba ed a 30'C in he da k. Cul u es used o gibbe ellin analyses we e g own in liquid medium. Those used o ca o enoid analyses we e g own on minimal aga o 4 days. Louis, Mo.) ha had been dissol ed in 0.2 ml o e hanol and mixed wi h esh medium and e hanolic sul u ic acid as desc ibed abo e. The de e mina ions o s anda d and p ob- lem samples we e un in pa allel. The alues gi en a e he a e ages o a leas wo de e mina ions. Rapid sc eening o gibbe eilin p oduc ion. The colonies o be sc eened we e ans e ed indi idually o ials wi h 1 ml o liquid medium and incuba ed o 9 days a 30°C. A 0.5-ml sample o cul u e medium was slowly added o a ube wi h 0.25 ml o sul u ic acid and obse ed unde a "black-ligh " lamp (Syl ania F18W/BLB/ES). The p esence o gibbe ellic acid in he sample led o a blue-g eenish luo escence. Mu agenesis. A wa e suspension o IM158289 spo es was exposed o 1 h a 22°C o 0.2 mg o N-me hyl-N'-ni o-N- ni osoguanidine pe ml (1), washed, and pla ed on minimal aga wi h 1 g o yeas ex ac pe li e . The esul ing colonies we e inocula ed wi h oo hpicks on o minimal aga , incu- ba ed o 3 days a 30°C, and hen ans e ed o liquid medium o he apid sc eening p ocedu e. The mycelia whose cul u e media showed educed o al e ed luo escence we e ans e ed o esh minimal aga and allowed o spo ula e o a epe i ion o he sc eening p ocedu e. RESULTS Simpli ied me hod o gibbe ellin analysis. The a ailable me hods o he analysis o gibbe ellin con en a e oo cumbe some o he quick sc eening o many samples. We de eloped a simpli ied me hod based on he luo escence o he eac ion p oduc s o gibbe ellins and sul u ic acid. An aliquo o he Gibbe ella cul u e medium is incuba ed wi h an e hanol solu ion o sul u ic acid. The ligh emission a 464 nm is de e mined in a spec o luo ime e a e exci a ion a 406 nm and compa ed wi h ha o a pu e gibbe ellic acid s anda d (Fig. 1). The emission and exci a ion luo escence spec a ob- se ed a e applica ion o his me hod o he cul u e media o he wild- ype IM158289 and he deep-o ange mu an SG22 coincided wi h ha o pu e gibbe ellic acid. The esul s o his me hod we e in good ag eemen wi h hose ob ained TABLE 2. Gibbe ellin p oduc ion in he wild- ype IMI58289 and in ca mu an s S ain Colo , da k/ligh Gibbe - G ow h (main ca o enoid)a (%)b (Y)C IM158289 Albino/O ange (NX) 100 100 Mu an s de i ed om IMI58289 SG2 Albino (none) 117 97 SG4 Albino (none) 118 99 SG53 Albino (none) 112 97 SG54 Albino (none) 97 92 SG43 Albino (phy oene) 108 98 SG1 Deep o ange (NX) 1 99 SG19 Deep o ange (NX) 55 94 SG22 Deep o ange (NX) 66 97 SG36 Deep o ange (NX) 54 95 SG37 O ange (NX) 44 95 SG39 Deep o ange (NX) 42 98 SG20 Pale o ange/o ange (NX) 62 107 SG48 Pale o ange/o ange (NX) 51 99 Mu an s de i ed om SG22 SG75 Albino (none) 45 90 SG76 Albino (none) 106 97 SG78 Albino (high phy oene) 70 98 SG68 Deep ed ( o ulene) 68 100 SG71 O ange (NX) 55 98 SG72 Pale o ange (NX) 110 97 SG73 Ve y deep o ange (NX) 46 96 a Acco ding o e e ence 3. Many s ains ha e he same colo a e g ow h in he ligh o in he da k. NX, neu ospo axan hin. b Gibbe ellins p esen in he cul u e medium a e 12 days o incuba ion ela i e o hose o he wild- ype con ols un in pa allel. The wild ype p oduced 76 ± 4 mg o gibbe ellins pe li e (mean and i s s anda d e o in 18 expe imen s). c Glucose consumed a e 12 days o incuba ion, ela i e o hose o he wild- ype con ols un in pa allel. The medium ini ially con ained 80 g glucose pe li e , and he wild ype consumed 31 ± 1 g (mean ± s anda d e o o 17 expe imen s). The wild ype eached 7.0 ± 0.2 g (d y weigh ) pe li e (10 expe imen s); he mu an s a e aged 6.6 ± 0.2 g (36 expe imen s). a e ex ac ion and sepa a ion o he gibbe ellins by high- pe o mance liquid ch oma og aphy (8). Isola ion o mu an s wi h al e ed gibbe ellin biosyn hesis. An e en simple sc eening p ocedu e was designed o he apid iden i ica ion o s ains wi h possible al e a ions in he p oduc ion o gibbe ellins. This me hod was applied o 4,100 colonies g own om spo es ha had been exposed o N-me hyl-N'-ni o-N-ni osoguanidine; 34 ga e educed o al e ed luo escence in h ee successi e sc eenings. A di e - ence om he wild ype was main ained by 14 isola es a e applica ion o he simpli ied analy ical me hod o he p e i- ous sec ion. These 14 isola es a e lis ed in Table 1. The h ee-le e code gib was chosen o he mu a ions espon- sible o modi ica ions in gibbe ellin biosyn hesis; he new s ains ca y he alleles gib-J h ough gib-14. Ca o enoid con en o he gib mu an s. All o he gib mu an s excep SG127 accumula ed ca o enoids in hei mycelia a 30°C in he da k (Table 2). The biosyn hesis o ca o enoids was pho oinducible in hese mu an s and in he wild ype: a 22°C hey accumula ed 45 o 111 ,ug o ca o - enoids pe g o d y weigh in he ligh bu less han 12 ,g/g in he da k. S ain SG127 was de oid o ca o enoids unde all condi ions es ed. These ca o enoid analyses we e ca ied ou wi h mycelia g own on minimal DG aga (1); hese mycelia p oduced no gibbe ellins. No co ela ion was seen VOL. 57, 1991 3380 CANDAU ET AL. J.5 LU U LU 0 LU GIBBERELLIC ACID (mg 1-1) FIG. 1. Fluo escence (emission a 464 nm a e exci a ion a 406 nm; means + s anda d e o s o wo independen de e mina ions) a e applica ion o he simpli ied analy ical me hod o s anda d solu ions o gibbe ellic acid. be ween he p oduc ion o ca o enoids and ha o gibbe el- lins by mycelia g own in liquid medium (Fig. 2). The mu an s SG133 and SG134 p oduce a mix u e o gibbe ellins ha is di e en om ha p oduced by he wild ype (7). The o he 12 gib mu an s p oduce less gibbe ellin han does he wild ype; he subs a es hus sa ed we e no di e ed in o ca o enoid p oduc ion (Fig. 2). Gibbe ellin p oduc ion in mu an s de ec i e o ca o eno- genesis. The wild ype can mu a e (3) o inc eased ca o eno- genesis (o ange, deep-o ange, and pale-o ange mu an s in he da k), o dec eased ca o enogenesis (albino mu an s 0.3 13$ E L 0.2 n a z UJ < 0.1 z a.. 0 0 An 0 10 20 30 40 GGPP IN GIBBERELLINS (jmol g-1) FIG. 2. Gibbe ellin and ca o enoid p oduc ion by he gib mu- an s a e 15 days o incuba ion a 30°C in liquid medium. To acili a e he compa ison, he con en s o bo h g oups o e penoids a e exp essed in e ms o he amoun (moles pe g am o mycelial d y weigh ) o he equi ed common p ecu so , ge anylge anyl py ophospha e. 150 CD E z 3 ILl TIME (days) FIG. 3. Gibbe ellin p oduc ion by s ains IMI58289 (wild ype), SG4 (no ca o enoids), SG22 (supe p oduce o neu ospo axan hin), and SG78 (supe p oduce o phy oene). wi hou ca o enoids), o o he same amoun o a di e en ca o enoid (phy oene). The ca mu an s di e in he p oduc- ion o gibbe ellins (Table 2 ahd Fig. 3). All o he inc eases in ca o enoid p oduc ion we e accompanied by dec eases in gibbe ellin p oduc ion. Th ee o ou mu an s ha lacked ca o enoids seemed o p oduce sligh ly la ge amoun s o gibbe ellins han did he wild ype. The phy oene-accumu- la ing mu an SG43 p oduced abou he same amoun o gibbe ellin as did he wild ype. The cul u e medium o one o he deep-o ange mu an s, SG1, was p ac ically de oid o gibbe ellins bu accumula ed a ed pigmen wi h an unknown chemical na u e. Mu an s wi h new colo changes we e de i ed (3) om one o he deep-o ange s ains, SG22. In some cases he gibbe - ellin p oduc ion emained low, bu i e u ned o he wild- ype le el in wo mu an s wi h dec eased ca o enoid p oduc- ion. The a ia ions in gibbe ellin p oduc ion canno be a ib- u ed o di e ences in g ow h, as shown by he analysis o he emaining glucose (Table 1). The e y la ge a ia ions in he con en o gibbe ellins and ca o enoids in di e en s ains app oxima ely compensa e each o he . In he s ains gi en in Fig. 4, he a ia ion coe icien s a e 109% o he ca o enoid con en s and only 12% o he sum o bo h g oups o e penoids. E ec on gibbe ellin p oduc ion o agen s ha a ec ca o- enogenesis. Ligh had no e ec on he p oduc ion o gibbe - ellins. This is shown in Fig. 5 o he wild ype unde condi ions ha no mally allow o ep ess gibbe ellin p oduc- ion and o s ain SG4, a whi e mu an unable o make ca o enoids ei he in he ligh o in he da k. Unde he same condi ions he e was no pho oinduc ion o ca o enogenesis: a e 15 days in liquid medium in he ligh o in he da k, he wild- ype mycelia con ained a o al o 24 o 29 ,ug o ca o enoids pe g o d y weigh . The wild ype does no lend i sel o he s udy o inhibi o s o ca o enogenesis because o i s low ca o enoid con en . The deep-o ange s ain SG22 had been used p e iously in i s place (3). a-Picoline inhibi s lycopene cycliza ion and delays 136 135 122 . 129 134 139 0 IM1I58289 * 128 0 0 121 * 133 0 124 .137 127 123 -. .d&. I APPL. ENVIRON. MICROBIOL. GIBBERELLINS AND CAROTENOIDS IN GIBBERELLA MUTANTS 3381 40- E %. 0. 0 30 20 - 10 - 0- IMI l58289 SG4 SG22 STRAIN FIG. 4. P oduc ion o gibbe ellins (=) and ca o enoids ( ) by wild- ype IM158289 and some ca o enoid mu an s a e 15 days o incuba ion a 30°C in liquid medium. The con en s o bo h g oups o e penoids a e exp essed in e ms o he amoun (moles pe g am o mycelial d y weigh ) o he equi ed common p ecu so , ge anylge- anyl py ophospha e. g ow h in G. ujiku oi. x-Picoline comple ely blocked gib- be ellin p oduc ion when added a a concen a ion o 2 g li e -' ei he be o e he inocula ion o he cul u es (Fig. 6) o a e gibbe ellin p oduc ion had s a ed (Fig. 7). Diphenylamine (20.3 mg li e -') and ,-ionone (200 mg li e -') inhibi ed g ow h when added be o e inocula ion o he cul u es. When hey we e added o 9-day-old cul- u es, he e was a pa ial inhibi ion o gibbe ellin accu- 1501 I cn z i Lu cm 100 50 TIME (days) FIG. 5. E ec o ligh on gibbe ellin p oduc ion. Symbols: 0, O, and A, cul u es g own in he ligh ; 0, *, and A, cul u es g own in he da k; Au and A, s ain SG4 g own in liquid medium; 0 and 0, wild- ype IM158289 g own in liquid medium; O and *, wild ype g own in liquid medium wi h 4.8 g o NH4NO3 pe li e . 6 LU 0 ao 0 LU 20 120 100 ab80 60 -I LU co' 40 20 20 IT 5 10 15 20 25 TIME (days) FIG. 6. E ec o a-picoline on he mycelial d y weigh (A) o s ain SG22 and on he glucose (B) and gibbe ellin (C) con en s o he medium. Symbols: 0, a-picoline (2 g li e -') added o he medium be o e inocula ion; 0, cul u es wi hou oL-picoline. mula ion (Fig. 7), which was p obably nonspeci ic because i was accompanied by a simila inhibi ion o g ow h. The conse ed luo escence spec a indica ed ha hese chemi- cals p oduced no g oss changes in he mix u e o gibbe el- lins. VOL. 57, 1991 -Vj 3382 CANDAU ET AL. Ox 100 E z LU s5 10 1S 20 25 TIME (days) FIG. 7. E ec on gibbe ellin p oduc ion by SG22 mycelia o a ious chemicals added o 9-day-old cul u es (a ow). Symbols: 0, cul u es wi hou chemical addi i es; A, emulsi ie s e hanol and Tween-80; *, a-picoline; *, diphenylamine; A, 13-ionone. DISCUSSION New me hods. The isola ion o he gibbe ellin mu an s and he o he esul s in his pape would ha dly ha e been possible wi hou he de elopmen o wo new me hods. The luo escence o he eac ion p oduc s o gibbe ellins wi h sul u ic acid is he ounda ion o adi ional analy ical me h- ods (14). Ou p ocedu e a oids he ex ac ion and pu i ica- ion s eps wi h hei concomi an losses o gibbe ellins. The p ocedu e migh gi e alse-posi i e eac ions wi h chemicals o he han he gibbe ellins. Tha his is no he case o ou Gibbe ella s ains is shown by he negligible luo escence obse ed wi h he wild ype in he p esence o a-picoline and wi h mu an s de oid o gibbe ellins. Ou apid sc eening p ocedu e may be used no only in he sea ch o mu an s bu in o he expe imen s as a apid i s es o iden i y he samples ha dese e p ecise analysis. Rela ionship be ween he biosyn heses o gibbe ellins and ca o enoids. S ain SG127 p oduced negligible amoun s o bo h e penoids. A single mu a ion could block he ans e o a p enyl esidue o amesyl py ophospha e o p oduce ge anylge anyl py ophospha e o shu o a possible common egula ion o bo h pa hways. The mu a ional incapaci y o p oduce amesyl py ophospha e should be le hal o lack o s e ols. The inc eased ca o enogenesis obse ed in he deep- o ange mu an s is quan i a i ely compensa ed by a de- c eased p oduc ion o gibbe ellins. The same seems o be he case, in he opposi e di ec ion, o he albino mu an s de oid o ca o enoids. These mu a ions do no a y he supply o p ecu so s bu di e hem o he gibbe ellin o he ca o - enoid b anch. The di e sion may occu a he le el o ge anylge anyl py ophospha e o any p e ious p ecu so . The albino mu an s may be unable o syn hesize phy oene om ge anylge anyl py ophospha e. The compensa ion ule does no apply o o he mu a ions, which seem o a ec only one o he pa hways. This is he case pa icula ly wi h mu a ions ha block he p oduc ion o gibbe ellins. A di e sion o he gibbe ellin p ecu so s in o he ca o enoid pa hway would esul in imp essi e pigmen- a ion le els (abou 10 mg g o d y weigh -'). Whi e illumina ion does no a ec he p oduc ion o gib- be ellin and ca o enoids in ou liquid cul u es. The ligh in ensi y used is no e y high bu is enough o b ing abou conside able pho oinduc ion o ca o enogenesis on aga cul- u es. a-Picoline blocks he biosyn heses o bo h neu ospo a- xan hin (2) and gibbe ellins in Gibbe ella. I does no p e en he p oduc ion o he common p ecu so ge anylge anyl py ophospha e, since neu ospo axan hin is eplaced by ly- copene. Thus, a-picoline inhibi s he o ma ion o he I8 ing o he ca o enoids and is likely o inhibi he cycliza ions in he gibbe ellin pa hway. Bo h pa hways include dehyd ogena ion s eps. Diphenyl- amine and ,-ionone inhibi he dehyd ogena ions om phy- oene o o ulene bu do no seem o ha e any speci ic e ec on gibbe ellin biosyn hesis. The h ee chemical inhibi o s s imula e he consump ion o glucose bu inhibi g ow h. They appea o uncouple he ca abolism o glucose om i s inco po a ion in o ungal biomass. ACKNOWLEDGMENTS We app ecia e he echnical assis ance o M. C. Vale o Qui 6s and J. C6 doba L6pez and he sugges ions and commen s o Edua do R. Beja ano and Pe e M. B amley. We app ecia e he inancial suppo o he Eu opean Commission (g an BAP-0395-E) and Comisi6n In e minis e ial de In es igaci6n Cien i ica y Tecnica (g an BT87-34). REFERENCES 1. A alos, J., J. Casadesus, and E. Ce da-Olmedo. 1985. Gibbe ella ujiku oi mu an s ob ained wi h UV adia ion and N-me hyl-N'- ni o-N-ni osoguanidine. Appl. En i on. Mic obiol. 49:187- 191. 2. A alos, J., and E. Ce dai-Olmedo. 1986. Chemical modi ica ion o ca o enogenesis in Gibbe ella ujiku oi. Phy ochemis y 25: 1837-1841. 3. A alos, J., and E. Ce da-Olmedo. 1987. Ca o enoid mu an s o Gibbe ella ujiku oi. Cu . Gene . 11:505-511. 4. A alos, J., and E. Sch o . 1990. Pho oinduc ion o ca o enoid biosyn hesis in Gibbe ella ujiku oi. FEMS Mic obiol. Le . 66:295-298. 5. Bea de , J. R. 1983. In i o di e penoid biosyn hesis in Gibbe - ella ujiku oi: he pa hway a e en -kau ene, p. 251-387. In A. C ozie (ed.), The biochemis y and physiology o gibbe ellins, ol. 1. P aege Publica ions, New Yo k. 6. B iickne , B., D. Blechschmid , G. Sembdne , and G. Schneide . 1989. Fungal gibbe ellin p oduc ion, p. 383-429. In E. J. Van- damme (ed.), Bio echnology o i amins, pigmen s and g ow h ac o s. Else ie Science Publishe s, L d., London. 7. Candau, R. 1991. P oducci6n de gibe elinas po Gibbe ella ujiku oi. Ph. D. hesis, Uni e sidad de Se illa, Se ille, Spain. 8. Candau, R., and P. B amley. Unpublished da a. 9. Dubois, M., K. A. Gilies, J. K. Hamil on, P. A. Rebe s, and F. S. Smi h. 1951. A colo ime ic me hod o he de e mina ion o suga s. Na u e (London) 168:167. 10. Geissman, T. A., A. J. Ve bisca , B. 0. Phinney, and G. C agg. 1966. S udies on he biosyn hesis o gibbe ellins om (-)- kau enoic acid in cul u es o Gibbe ella ujiku oi. Phy ochem- is y 5:933-947. 11. Goodwin, T. W. 1980. The biochemis y o he ca o enoids, ol. 1, 2nd ed. Chapman and Hall, London. 12. Je e ys, E. G. 1973. The gibbe ellin e men a ion. Ad . Appl. Mic obiol. 13:283-315. 13. Pha is, R. P., and R. W. King. 1985. Gibbe ellins and ep oduc- i e de elopmen in seed plan s. Annu. Re . Plan Physiol. 36:517-568. 14. Takahashi, N., I. Yamaguchi, and H. Yamane. 1986. Gibbe el- lins, p. 57-151. In N. Takahashi (ed.), Chemis y o plan ho mones. CRC P ess, Boca Ra on, Fla. APPL. ENVIRON. MICROBIOL.