APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1990, p. 2303-23100099-2240/90/082303-08$02.00/0Copyright ©D 1990, American Society for Microbiology

Vol. 56, No. 8

Spore Yield and Microcycle Conidiation of Colletotrichumgloeosporioides in Liquid Culture

J. J. CASCINO,1 R. F. HARRIS,2* C. S. SMITH,3t AND J. H. ANDREWS3Departments of Botany,' Soil Science,2 and Plant Pathology,3 University of

Wisconsin-Madison, Madison, Wisconsin 53706

Received 5 December 1989/Accepted 10 May 1990

The effect of V8 juice concentration (5 to 40%, vol/vol), spore inoculum density (105 and 107 spores per ml),and liquid batch or fed-batch culture condition on mycelium and spore production by Colletotrichumgloeosporioides was evaluated. The amount of mycelium produced, the time required for initiation ofsporulation following attainment of maximum tnycelium, and the time for attainment of maximum spore

concentration increased with increasing V8 juice concentration in batch culture. Cultures containing V8 juiceat >10% achieved a similar spore density (apparent spore-carrying capacity) of about 0.8 mg of spores per ml(1 X 107 to 2 x 107 spores per ml) independent of inoculum density and V8 juice concentration. The relativespore yield decreased from a high of 64% of the total biomass for the low-inoculum 5% V8 culture, through13% for the analogous 40% V8 culture, to a low of 2% for the high-inoculum 27% V8 culture. Fed-batchcultures were used to establish conditions of high spore density and low substrate availability but high substrateflux. The rate of addition of V8 juice was adjusted to approximate the rate of substrate utilization by the(increasing) biomass. The final spore concentration was about four times higher (3.0 mg of spores per ml) thanthe apparent spore-carrying capacity in batch culture. This high spore yield was obtained at the expense ofgreatly reduced mycelium, resulting in a high relative spore yield (62% of the total biomass). Microcycleconidiation occurred in the fed-batch but not batch systems. These data indicate that substrate-limited,fed-batch culture can be used to increase the amount and efficiency of spore production by C. gloeosporioidesby maintaining microcycle conidiation conditions favoring allocation of nutrients to spore rather thanmycelium production.

Increasing attention is being paid to the use of microor-ganisms in biological control of pathogens, weeds, andinsects (7, 10, 11, 15). The fungus Colletotrichum gloeo-sporioides has been successfully used to control severalspecies of weeds (6, 8, 9, 28). Strains of this fungus havebeen patented (J. T. Daniel, G. E. Templeton, and R. J.Smith, Jr., U.S. patent 3,849,104, Nov. 1974) for the controlof northern jointvetch (Aeschynomene virginica) and wingedwaterprimrose (Jussiaea decurrens), serious weeds in soy-bean and rice fields, respectively. More recently, C.gloeosporioides has been suggested as a possible biologicalcontrol agent for Clidemia hirta, an invading weed in Ha-waiian forests (29).

Spores are the most common propagule used in biocontrolprograms (8) and must be produced in large quantitiesquickly, inexpensively, and efficiently (15). Fungal sporesare normally mass produced in large liquid culture fermen-tations (8), and information on the effects of manipulatingliquid culture conditions to maximize the efficiency of sporeproduction is of potential practical value. Reduction ofmycelium in liquid culture would also be desirable, since itcreates separation and disposal problems.There is limited information on specific growth yields

(mass of biomass produced/mass of limiting substrate added)and the proportion of total biomass produced as spores(defined here as relative spore yield). Daniel et al. (9)reported good growth and sporulation in lima bean agar and

* Corresponding author.t Present address: Environmental Laboratory ER-A, Waterways

Experiment Station, Vicksburg, MS 39181.

in a liquid medium composed of V8 juice, sucrose, nitrate,and mineral supplements. According to Churchill (8), C.gloeosporioides grows well in several media, but precisebalancing of the levels of nitrogen, carbon, and mineralsupplements is required for optimal sporulation. Lingappaand Lingappa (13) found that increasing concentrations ofsucrose caused preferential development of mycelia. Sladeet al. (23, 24) ranked the solid and liquid forms of 11 commonmedia for spore production and found that V8 juice with nosupplements and an organic concentration of 6 mg/ml (15%unfiltered V8 juice) was optimal for high sporulation and lowmycelium production.The proportion of spores to total biomass (relative spore

yield) is potentially a function of the concentration of regu-lating metabolites. C. gloeosporioides produces a self-inhib-itor of spore germination, gloeosporone (12, 14); metabolicinhibition of sporulation by C. gloeosporioides at high sporeconcentrations may contribute to an apparent spore-carryingcapacity (SCC), independent of nutrient level, shown bybatch cultures of this organism on relatively concentratedsolid media (23). Relative spore yields of C. gloeosporioidesmay also be a function of aeration levels, with increased 02levels producing higher or lower relative yields depending onthe pathovar (8).High relative spore yields are favored by microcycle

conidiation. Microcycle conidiation is defined as sporulationdirectly after spore germination, with greatly reduced or nomycelial growth (26). It can be induced by high temperatures(1, 2, 21), nutrient depletion (5, 16), and/or other factorswhich inhibit vegetative growth (17, 20, 23). Work withGlomerella cingulata (the teleomorph of C. gloeosporioides)(13) and C. gloeosporioides (23) demonstrated that on solid

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2304 CASCINO ET AL.

media a high density conidial inoculum resulted in yeastlikegrowth (microcycle conidiation), characterized by acceler-ated development of short conidiophores supporting a gelat-inous mass of slime spores (4, 23). However, for C.gloeosporioides, such microcycle induction was dependenton the medium concentration, since concentrations of inoc-ulum (2 x 106 spores per ml) which induced microcycleconidiation on solid media containing up to 20% (vol/vol) V8juice produced extensive mycelial growth when applied tothe same solid media containing higher concentrations of V8juice (23). Microcycle-inducing concentrations of inoculumdid not increase the maximum spore concentration producedby C. gloeosporioides on solid media, but did shorten thetime taken to reach it. Slade et al. (23) hypothesized thatmicrocycle conidiation was induced by buildup of a metab-olite, nutrient limitation, or both.Work with G. cingulata in liquid culture indicated that an

inoculum sufficient to induce microcycle conidiation in liquidculture increases the relative spore yield and the final sporeconcentration (13). More recent work with deep-tank fer-mentations (experimental conditions unidentified) showedthat cultures of C. gloeosporioides undergoing microcycleconidiation can produce almost 100% of the total biomass asspores (8).We describe here, for liquid batch cultures of C. gloeospo-

rioides growing in V8 juice medium (a preferred medium forthis organism [24]), the effect of initial V8 concentration andinoculum density on (i) the time course for mycelium andspore production and decay, (ii) the efficiency of substrateconversion to fungal biomass (specific yield), and (iii) therelative efficiency of spore production. In addition, wereport that the apparent SCC shown by this organism inbatch culture can be exceeded in substrate-limited fed-batchculture and that the microcycle conidiation phenomenonobserved on solid media for this organism can be inducedand maintained in an analogous (from a substrate supply andhigh-spore-density standpoint) fed-batch liquid system.

MATERIALS AND METHODS

Inoculum source. A strain of C. gloeosporioides wasoriginally isolated from a diseased watermilfoil plant (Myrio-phyllum spicatum) in Madison, Wis., and stored on silica gelcrystals (18). Working cultures were maintained on peptoneglucose agar (30) at 25°C in natural light. Conidia for exper-imental inocula were obtained by growing the fungus in 100ml of 20% (vol/vol) V8 juice (Campbell Soup Co., Camden,N.J.) on a rotary shaker at 250 rpm and 25°C as describedpreviously (23, 24). After 4 days, the mycelium was removedby filtration through two layers of sterile cheesecloth, andthe spores were washed three times by centrifugation andsuspension in sterile deionized water before use.Media. For the batch experiments, V8 juice was filtered

through two to four layers of cheesecloth and diluted to 5,10, 20, and 40% concentrations in deionized water. Todetermine the solids content, duplicate 35-ml samples ofeach dilution of filtered V8juice were placed in a preweighedaluminum pan and dried at 65°C to constant weight (2 days).Filtration was needed to minimize solid-particle interferencewith biomass dry-weight measurements and to preventblockage of the small-diameter tubing used in the fed-batchsystems.

Experimental procedure for batch culture. Two kinds ofbatch culture studies were done: low-inoculum and high-inoculum experiments. In the low-inoculum experiment,

125-ml Erlenmeyer flasks containing 50 ml of 5, 10, 20, or40% filtered V8 juice and an initial inoculum concentration of105 spores per ml were incubated at 25°C on a rotary shakerat 250 rpm in natural light. Triplicate flasks were harvested at1, 2, 3, 4, 5, 6, 8, 12, and 16 days in the long-termexperiments and at 6, 12, 18, 24, 32, 40, 48, 60, 72, 84, and 96h in the short-term experiments. The experiment was con-ducted twice for each V8 concentration. In the high-inoc-ulum experiment, batch cultures consisted of 125-ml Erlen-meyer flasks containing 40 ml of 40% V8 juice and 20 ml ofa 4-day-old (organic substrate-spent) 10% V8 juice culturefrom which the mycelium had been removed by filtration andthe spore concentration had been adjusted to give an initialspore density (60-ml volume basis) of about 107 spores perml. Three replicate flasks were incubated for 2 days andharvested as described for the low-inoculum experiments.Three independent runs were done for this experiment.Each flask was harvested by filtering the contents through

a preweighed double layer of cheesecloth under vacuum toseparate the harvestable spores from the mycelium. Eachcheesecloth filter was washed once with 50 ml of deionizedwater. Spores removed from the filters by this wash (inpractice quantitatively essentially negligible) were combinedwith their original spore suspensions and centrifuged at12,000 x g for 6 min. The resulting pellets were washed threetimes by suspension in 50-ml volumes of deionized water andrecentrifugation. The number of spores in each filtrate wasdetermined (25) by counting 25 fields of view (0.2 by 0.2 mm)from each side of a Reichert Brightline hemacytometer(Warner-Lambert Technologies Inc., Buffalo, N.Y.). Fordry weight yields, the spore suspensions were collected byvacuum filtration onto tared fiber glass filters (934AH grade;Reeve Angel, Clifton, N.J.). All preweighed cheesecloth andfiber glass filters were washed in deionized water and driedat 65°C for 2 days prior to tare determination. The mycelium-containing cheesecloth and spore-containing fiber glass fil-ters were dried at 65°C for 2 days, and dry weights werecalculated by difference. Since spore dry weight varies for C.gloeosporioides depending on culture age (25), it was neces-sary, apart from elimination of accuracy losses resultingfrom the use of a conversion factor, to weigh spores directlyrather than calculate spore dry weight yield from sporenumbers.

Prior to flask sacrifice for biomass harvesting, a 1-mlsubsample was removed for qualitative microscopic exami-nation for microcycle conidiation as evidenced by primaryconidia formed via budding or binary fission, or secondaryconidia produced on the ends of short germ tubes of themother conidium (8).

Experimental procedure for fed-batch culture. Fed-batchexperiments were conducted in 1-liter VirGlas (The VirTisCo., Inc., Gardiner, N.Y.) sidearm flasks stirred by a built-inmagnetic impeller. A peristaltic pump (Gilson Medical Elec-tronics, Inc., Middleton, Wis.) was used to supply 40% V8juice from a 3-liter reservoir flask to the medium input tubeof the VirGlas culture via a hypodermic needle. The V8 juicemedium was forced into the culture by introducing sterile airat 400 ml/min through a second needle in the medium inputtube. To prevent foaming, 0.05% Antifoam A (Sigma Chem-ical Co., St. Louis, Mo.) was added to the starting volume.To achieve substrate-limited conditions, the rate of substrateaddition was adjusted to be substantially lower than theapproximated maximum rate of substrate demand (see Ap-pendix).Two fed-batch conditions were established: severely sub-

strate limited (one replicate) and marginally substrate limited

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SPORE YIELD AND MICROCYCLE CONIDIATION 2305

(three replicates run at different times). The severely sub-strate-limited fed-batch culture initially contained about2 x 107 spores per ml (1.25 mg of spores per ml) in 300 mlof a 4-day-old (substrate-spent) 10% V8 juice culture fromwhich the mycelium had been removed. Initially, 40% V8juice (20.5 mg of V8 solids per ml) was added at 0.8 mlIh,increasing the volume from 300 to 319 ml over the first24 h. From 24 to 48 h, the rate was increased to 2.4 ml/hto account for the increase in biomass (and consequentincrease in substrate demand by the biomass), therebyincreasing the volume from 319 to 377 ml by the end of thesecond day. The final amount of fresh V8 juice solids addedwas comparable to a 377-ml batch system initially contain-ing (24 x 0.8 x 20.5 + 24 x 2.4 x 20.5)/377 = 4.2 mg ofV8 solids per ml. The marginally substrate-limited fed-batch culture contained about 1 x 107 spores per ml (0.4mg of spores per ml) in 200 rather than 300 ml of spent 10%V8 juice medium. The initial input rate of 40% V8 juice (21.1mg of V8 solids per ml) was 4.17 ml/h, increasing the volumefrom 200 to 300 ml over the first 24 h; from 24 to 48 h, theinput rate was increased to 12.5 ml/h, increasing the volumefrom 300 to 600 ml by the end of the second day. The finalamount of fresh V8 juice solids added was comparable to a60-ml batch system initially containing 14.1 mg of V8 solidsper ml.Each fed-batch culture was sampled periodically for spore

counting and observations of microcycle conidiation (as forthe batch systems) by removing 10 ml of the culture asepti-cally with a wide-mouthed pipette. At the final harvest, dryweights and spore counts of triplicate 50-ml subsampleswere determined in the same manner as for the batchexperiments.

Data analysis. To determine the period when the spore dryweight yield (concentration) was at its maximum for eachconcentration of V8 juice in the batch experiments, weanalyzed the spore concentration at different sampling timesby an analysis of variance followed by a Duncan multiple-range test (27) by using the SAS analysis of varianceprocedure (22). All of the samples in the Duncan group withthe highest spore concentrations were considered to repre-sent samples at the maximum spore yield. Averages ofbiomass values from cultures in this group were used torepresent spore and mycelium yields during the period whenthe cultures had reached the maximum spore yield. Althoughmycelium yields declined with time during this maximizedspore density period, averages are useful for comparingdifferent V8 juice concentrations.Biomass and spore yields (Table 1) were calculated as

follows.

Spore yield (YSP):Ysp = (final spore concentration x final volume) -

(initial spore concentration x initial volume)final volume

Specific total biomass yield (YI,,):Y,1, = (final dry weight concentration x final volume) -

(initial dry weight concentration x initial volume)concentration of V8 solids x final volume

Specific spore yield (Ys,,P):Ysp/, = (final spore concentration x final volume) -

(initial spore concentration x initial volume)concentration of V8 solids x final volume

Relative spore yield (RSY):RSY = (YSP/SYX/S) x 100

RESULTSLow-inoculum batch culture. The effect of initial V8 juice

concentration on the time course for mycelium and sporeproduction and degeneration is illustrated in Fig. 1 for thelong-term (16-day) experiments. Figures 2 and 3 depict theshort-term (4-day) experiments focusing on the shift frommycelium to spore production. Experimental conditions andbiomass concentrations and growth yields for the periodshowing the maximum dry weight-based spore yields aresummarized for all experiments in Table 1. Similar trendswere shown for all concentrations of V8 juice tested: (i) theinitial growth phase (increasing total biomass) was confinedto mycelium production; (ii) mycelium production essen-

tially ceased once sporulation was initiated; (iii) there was a

relatively short phase with increasing spore and decreasingmycelium concentration until a constant maximized sporedensity was achieved; and (iv) total biomass declined rela-tively slowly during the following period of maximized sporedensity, largely as a function of mycelium loss.The duration of the mycelium production phase, and the

concentration of mycelium during the period of maximizedspore density, increased in proportion to increasing V8 juiceconcentration (Fig. 1; Table 1). For example, the concentra-tion of mycelium during the period of maximized sporedensity increased from a low of 0.3 to a high of 5.4 mg ofmycelium per ml for the 5 and 40% V8 juice concentrationsystems, respectively. In marked contrast, the spore densityshowed an apparent SCC of about 0.8 mg of spores per mlindependent of V8 juice concentration above 10%. Becauseof the negligible quantitative contribution of the inoculum,the spore yield (Ysp) was the same numerically as the sporeconcentration for the low-inoculum systems. The biomassconcentration data were transformed into specific (per unitof V8 juice solids) growth yields. The specific total biomassyield (YX/S) was relatively constant (about 0.3 mg/mg of V8solids) over the range of V8 juice concentrations tested. Theprogressive increase in mycelium concentration but rela-tively constant spore concentration with increasing V8 juiceconcentration gave rise to a progressive decline in thespecific and relative spore yields (Table 1). For example, therelative spore yield decreased from a high of 64% for the 5%V8 juice batch culture to 13% for the 40% V8 juice system.No microcycle conidiation was observed at any time in

any of the low-inoculum batch cultures. Conidiophores anda few conidia were observed earlier than sporulation was

detected by spore dry weights.High-inoculum batch culture. Initial spore concentrations

were adjusted to be similar to the final spore concentrations(apparent SCC) shown by the low-inoculum batch cultures(Table 1). Spore concentrations declined during the phase ofspore germination and mycelial growth (first day) and thenmarginally regained their initial concentration (apparentSCC) following the sporulation phase (day 2); flasks incu-bated for an additional 2 days did not show further increasesin spore concentration (data not shown). Mycelial produc-tion was high (3.9 mg/ml), and specific biomass yield was

similar to that shown by the low-inoculum batch cultures(0.3 mg/mg of V8 solids). The spore mass was substantiallyless (0.03 mg/106 spores) than in typical batch culture (0.08 to0.1 mg/106 spores). Because the initial and final spore dryweight concentrations were almost the same, the specificand relative spore yields were the lowest obtained in any

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SPORE YIELD AND MICROCYCLE CONIDIATION 2307

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TIME (days)

FIG. 1. Effect of V8 juice concentration on mycelium and spore

dry weight kinetics in liquid batch culture inoculated with a low-density spore inoculum (105 spores per ml, <0.01 mg of spores per

ml) and incubated for 16 days (long-term experiments). Symbols for5% V8 juice: 0, mycelium; *, total; for 20% V8 juice: A, mycelium;A, total; and for 40% V8 juice: El, mycelium; *, total. The shadedareas between the total and mycelium lines represent the spore

concentration. Bars indicate + 1 standard deviation. Where errorbars are not shown, they are contained within the symbol.

system. Some individual flasks had a final spore dry weightconcentration less than that of the initial inoculum. Veryhigh inoculum density (e.g., >5.6 mg of spores per ml) batchcultures similar to those used for G. cingulata (13) were notevaluated because of the routine impracticality of suchmassive inoculum systems. No microcycle conidiation wasobserved at any time in our high-inoculum batch cultures.

Fed-batch culture. The effect of continuous addition of40% V8 juice on the mycelium and spore concentrations andyields in fed-batch cultures initially containing spore concen-trations of the same order of magnitude as the apparent SCCin batch culture is presented in Table 1. Under severelysubstrate-limited conditions, the spore density reached 2.7mg of spores per ml at the end of the experiment (2 days);

1.2-

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FIG.E 2. Mycelium and spore dry weightkinetics.in.5%.V8.juiceli 0.6 culture.....ulated...tha low-density spore. inoculum(105 spores per ml, <0.01 mg of spores perml).and. incubated.for. 4

days(short-term experiment)....Sy.mbols:..........................tal..

Shaded area as.in.Fig..1...

~~ ~ ~ ~ ~ ~ ~ ~

..

s).. ..

.. .. ..

TIME (hours)FIG. 3. Mycelium and spore dry weight kinetics in 20% V8 juice

liquid batch culture inoculated with a low-density spore inoculum(105 spores per ml, <0.01 mg of spores per ml) and incubated for 4days (short-term experiment). Symbols: A, mycelium; A, total.Shaded area as in Fig. 1.

this substantially exceeded the apparent SCC of about 0.8mg spores per ml shown in batch culture. The efficiency ofsubstrate conversion to biomass was very high, and 80% ofthe biomass produced was spores, a relative spore yieldhigher even than that shown by the 5% V8 juice batchculture.Under the marginally substrate-limited conditions, the

spore density reached 3.1 mg of spores per ml at the end ofthe experiment (2 days), similar to that shown under severesubstrate limitation and three times higher than the highestspore density obtained in batch culture (Table 1). Myceliumproduction was higher than under severe substrate limitation(1.9 versus 0.4 mg of mycelium per ml) but was much lowerthan for the analogous batch culture with 14.1 mg of V8solids per ml (3.5 mg of mycelium per ml; estimate fromTable 1). The efficiency of substrate conversion to biomasswas comparable to that in the batch cultures. The specificand relative spore yields for the marginally substrate-limitedfed-batch culture were similar to the analogous, relativelyhigh yields for the 5% V8 juice batch culture, but because ofthe much higher level of V8 juice addition (14.1 versus 2.5mg of V8 solids per ml), the spore yield was much higher forthe fed-batch culture (3.0 versus 0.6 mg of spores per ml).

Spores from the fed-batch cultures were observed to beundergoing microcycle conidiation. All three forms of micro-cycle conidiation previously reported (8) for C. gloeospon-oides were apparent: primary conidia were formed viabudding and binary fission, and secondary conidia wereproduced on the ends of short germ tubes of the motherconidium. The percentage of spores coming from each formof microcycle conidiation was not quantified, but secondaryconidiation and binary fission were very common, whereasbudding was relatively rare.

DISCUSSION

Our results demonstrate the importance of substrate avail-ability kinetics as a factor determining maximum sporeconcentrations and specific and relative spore yields ofC. gloeosporioides in liquid culture.

Batch culture growth and interpretation of the existence ofan apparent SCC in batch systems. The proportionate in-crease in total biomass with increasing V8 juice concentra-

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2308 CASCINO ET AL.

tion (Fig. 1), as shown by the constant specific total biomassyield of about 0.3 mg of cells per mg of V8 solids per ml(Table 1), indicates that in all batch systems, growth cessa-tion was due to substrate limitation rather than productinhibition. Growth in batch culture was initially mainlymycelial, and the onset of sporulation corresponded closelyto reduction (Fig. 2) or cessation (Fig. 1 and 3) of growth.This indicates that as long as V8 substrates were available atnonlimiting levels, they were used by C. gloeosporioides formycelial growth. For batch cultures of 5 and 10% V8concentration, the spore yield was limited to some extent bythe initial amount of available substrate present in thesesystems (Table 1).For batch systems with a V8 concentration higher than

10%, the spore yield was relatively constant, independent ofthe initial spore inoculum density and the stationary-phaseconcentration of mycelium (Table 1). The latter phenomenongave rise to an apparent SCC of about 0.8 mg of spores perml in batch cultures containing a relatively high V8 concen-tration; this is consistent with earlier findings for this organ-ism on solid media (23). The time course data for the 20% V8juice system (Fig. 3) show that following biomass maximi-zation exclusively in the form of mycelium, production ofspores was accompanied by a decrease in the amount ofmycelium present. This suggests that sporulation wasachieved at least in part by use of endogenous material. Twodifferent endogenous sporulation-based mechanistic expla-nations of the apparent SCC phenomenon are possible. Thefirst of these (metabolite inhibition mechanism) is thatendogenous sporulation was inhibited by metabolites pro-duced by spores once a maximized spore density wasachieved. The second mechanistic explanation (competent-mycelium mechanism) is that endogenous sporulation couldarise only from young (competent) mycelium and that theamount of such mycelium was relatively constant for all V8juice concentrations in batch cultures at the initiation ofavailable substrate-limitation. Trial experiments (data notshown), in which washed mycelia from SCC cultures weresuspended in inorganic media, showed that endogenoussporulation was not induced; also, when minimally washedmycelia obtained from substrate-unrestricted cultures weresuspended in spore-free, SCC spent media, mycelial sporu-lation was not prevented. These findings do not support themetabolite inhibition explanation of batch culture SCC.Furthermore, whereas the metabolite inhibition mechanismpredicts that the existence of a true SCC would impose aceiling on the achievable spore density under all cultureconditions, the competent-mycelium mechanism predictsthat the apparent SCC should be overcome under conditionsof continually growing mycelium exposed to a finite butgrowth-limiting (sporulation-inducing) supply of organic sub-strate. The fed-batch data showing spore densities three- tofourfold higher than those in batch culture support thecompetent-mycelium rather than metabolite inhibition mech-anism for explaining the apparent SCC observed in batchsystems.

In summary, it appears that as long as the organic sub-strate is nonlimiting, C. gloeosporioides growth is mainly, ifnot exclusively, mycelial. As soon as the substrate poolbecomes limiting, spore production is initiated by myceliausing residual substrate and/or endogenous resources. Sporeproduction then proceeds until the residual substrate iscompletely depleted and/or the potential of the competentmycelium for endogenous sporulation is completely realized.

Microcycle conidiation and spore yield in fed-batch culture.In accordance with the above substrate availability-based

mechanism of sporulation control, continuous sporulationvia microcycle conidiation would be expected to occur ifsubstrates were added continuously to a C. gloeosporioidespopulation at a rate such that the substrates were consumedas fast as they were added. This would provide a continuoussubstrate supply for sustained growth but maintain a sub-strate level low enough to induce sporulation. The sporeconcentration in such a system should increase above theobserved ceiling in batch culture as long as the buildup ofmetabolites does not limit growth or sporulation; the exist-ence of a high spore density might also contribute to micro-cycle conidiation via the production of metabolic promoters,although direct evidence of such a phenomenon is notcurrently available. Our fed-batch cultures were initiatedwith a high spore density as a convenient experimentalstarting condition and were established to meet the require-ments for a continuous flux of substrates through a limitingsubstrate pool, by adjusting the rate of substrate input to belower than the approximated maximum (substrate-unlimited)rate of substrate demand by the fungal population (seeAppendix). Consistent with projections, growth was domi-nated by microcycle conidiation and spore concentrationsexceeded (three- to fourfold) those in batch culture (Table 1).The severely substrate-limited fed-batch culture producedrelatively much less mycelium and consequently gave amuch higher relative spore yield (80 versus 62%) than themarginally substrate-limited fed-batch system (Table 1), butat the expense of reduced spore production (1.7 mg of sporesper ml in 377 ml versus 3.0 mg of spores per ml in 600 ml). Ona commercial scale, the high relative spore yield of theseverely substrate-limited system would have to be weighedagainst the added time it would take for production of thedesired biomass of spores.

This work demonstrates that spore yields greater than thepreviously reported maximum in batch culture can be at-tained in substrate-limited, fed-batch culture. Spores in suchfed-batch systems undergo microcycle conidiation, and this,in effect, causes a diversion of nutrients from mycelium tospore production. Such systems would appear to havepotential for commercial spore production by C. gloeospo-rioides and similar fungi showing substrate availability-dependent mechanisms of microcycle conidiation.

APPENDIXThis appendix contains calculations for establishment of sub-

strate-limiting conditions in fed-batch culture. The rate of substrateaddition, R, (milligrams of substrate per hour), was adjusted to beless than the approximated maximum rate of substrate demand,Q, (milligrams of substrate per hour) to achieve substrate-limitingconditions. Rs was derived as follows:

R, = RxX Cso (Al)where R, is the rate of substrate (V8 juice) addition (milliliters perhour) and Cso is the influent substrate concentration (milligrams ofsubstrate per milliliter). Qsmax was derived as

(A2)Qsqs m ax X

= (max I Yx/s) x (C,, x V)where qx is the maximum specific rate of substrate demand(milligrams of substrate per milligram of biomass per hour), Uma" isthe substrate-unlimited specific growth rate (reciprocal hours), YX/Sis the specific growth yield (milligrams of biomass per milligram ofsubstrate) at Ilmax, X is the amount of biomass in the culture(milligrams), C, is the biomass concentration (milligrams per milli-liter), and V is the volume of the culture (milliliters) (3, 19). Table 1gives values for R,, C,0, V, and C, for different fed-batch systems.From Fig. 2 and 3, the biomass doubling time is in the 10- to 3-h

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range, which translates into, recognizing that Ilmax = ln(2)/doublingtime, a jmax range of 0.07 to 0.23 h-'. This gives, for YX,/ = 0.3 mgof biomass per mg of substrate (Table 1), a qsmax range of (fromequation A2)

qSmax = (ilmax I Yx/s) (A3)= (0.07 to 0.23)/0.3

= 0.2 to 0.8 mg of substrate per mg of biomass per hTo obtain severely substrate-limited conditions, Rs was conser-

vatively adjusted (using qs = 0.2 mg of substrate per mg ofbiomass per h) to be less than the lowest level of the approximatedQ5 during the fed-batch experiment. At 0 h, Rs = 16 < Q5 = 75to 9T0 mg of substrate per h [calculated from equation Al anWdTable1, Rs = RV x Cs = 0.8 x 20.5 = 16; and from equations A2 and A3and Table 1, Q5 =q5 x X = q5 x (C5X V) = (0.2 to 0.8) x(1.25 x 300) = / to 306T.At 24 h, Rs = 16(( Q5=99to394mgof substrate per h {Rs was constant from 0 to 24 h; and fromequations A2 and A3 and Table 1, recognizing that the zerosuperscript denotes a value at time 0 h, Q = qs x [Xo + DX] =

q x [(C_° x VW) + (t x R x Cs. x Yx5 = (0m. to 0.8) x [(1.25xm'00) + (24 x 0.8 x 20.5 x 0.3)] = 99 to 394}. Immediatelyfollowing the R, upshift at 24 h, Rs = 49 < Qsmax = 99 to 394 mg ofsubstrate per h (calculated from equation Al and Table 1, Rs =Rx CSO = 2.4 x 20.5 = 49; and Q5 was unchanged). At 48 h, Rs =

49 << Q, = 241 to 965 mg of'substrate per h [Rs was constantfrom 24 to 48 h; and from equations A2 and A3 and Table 1, Q5 =

q x (C, x V) = (0.2 to 0.8) x (3.2 x 377) = 241 to 965].Accordingly, the substrate limitation requirement for substratesupply to be less than substrate demand was more than adequatelymet throughout the entire 48-h period for the severely substrate-limited fed-batch culture.A similar approach was taken to establish marginally substrate-

limited conditions. However, to maximize biomass productionkinetics, the input rates of fresh media (Rs) were adjusted to besimilar to the approximated substrate demand rates (Q5 ) at 12 h(rather than 0 h) and 36 h (rather than 24 h). At 0 h, Rs =% > Q,5= 16 to 64 mg of substrate per h [calculated from equation Al andTable 1, Rs = R, x Cs = 4.17 x 21.1 = 88; and from equations A2and A3 and Table 1, Q5. = q X X =q5 x (C, X V) = (0.2 to0.8) x (0.4 x 200) = 16 to 64]. At 12 h, Rs-=8 versus Q5 = 79 to317 mg of substrate per h {Rs was constant from 0 to 24 h; and fromequations A2 and A3 and Table 1, Qs = q x [Xo + DX] = qx [(CxO x VW) + (t x R, x Cs. x YX7 = (0.2 to 0.8) x [(0.4 x 200)+ (12 x 4.17 x 21.1 x 0.3)] = 79 to 317}. At 24 h, Rs = 88 < Q5= 143 to 571 mg of substrate per h {R, was constant from 0 to 24 rand from equations A2 and A3 and Table 1, Qs = qsm. x [(CxO xW) + (t x R, x Cso x YXS] = (0.2 to 0.8) x [(0.4x 200) + (24 x 4.17x 21.1 x 0.3)] = 143 to 571}. Immediately following the Rs upshiftat 24 h, Rs = 264 versus Qs = 99 to 394 mg of substrate per h(calculated from equation Al and Table 1, Rs = R, X Cs. = 12.50 x

21.1 = 264; and Q5 was unchanged]. At 36 h, Rs = 264 < Qsm =333 to 1,330 mg of substrate per h {Rs was constant from 24 to 48 h;and from equations A2 and A3 and Table 1, Qsm = qsma5 x [(CX0 xV)+ (t x R x Cso x Yx1s)] = (0.2 to 0.8) x [(0.4 x 200) + ((24 x4.17) + (12 x 12.5)) x 21.1 x 0.3)] = 333 to 1,330}. At 48 h, Rs = 264(( Q = 600 to 2400 mg of substrate per h [Rs was constant from 24to 48 h; and from equations A2 and A3 and Table 1, Qs = q5 x

(Cx x V) = (0.2 to 0.8) x (5.0 x 600) = 600 to 2,400]. Accordingly,the substrate limitation requirement for Rs < Q5 was not met at alltimes during the incubation, particularly at time mOi and immediatelyfollowing the Rs upshift at 24 h. However, at 24 h and at the end ofthe experiment (48 h), the Rs < Qsax requirement was more thanadequately met.

ACKNOWLEDGMENTSThis research was supported by the College of Agricultural and

Life Sciences, University of Wisconsin-Madison, by U.S. ArmyCorps of Engineers (Aquatic Plant Control Research Program)contract DACW39-86-K-0020, and by Environmental ProtectionAgency grant R-811587-01-0.We thank S. Slade for preliminary experimental and conceptual

contributions and P. Cavey and L. Schille for technical assistance.

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