Biological Control 39 (2006) 232–239 www.elsevier.com/locate/ybcon
The eVect of cane molasses amendment on biocontrol of frosty pod rot (Moniliophthora roreri) and black pod (Phytophthora spp.) of cocoa (Theobroma cacao) in Panama
Ulrike Krauss a,¤, G. Martijn ten Hoopen b, Eduardo Hidalgo a, Adolfo Martínez a, Tim Stirrup c,1, Claudio Arroyo a, Johnny García a, Manuel Palacios d
a CABI-CATIE-USDA, Centro Agronómico de Investigación y Enseñanza (CATIE), 7170 Turrialba, Costa Rica b CABI-CATIE-DGIS, CATIE, 7170 Turrialba, Costa Rica c Department of Natural Sciences, University of Bath, Bath BA2 7AY, UK d Cooperativa Cacaotera Bocatoreña (COCABO), Almirante, Bocas del Toro, Panama
Received 27 February 2006; accepted 12 June 2006 Available online 14 June 2006
Abstract
Frosty pod rot (FPR), caused by Moniliophthora roreri, and black pod (BP), caused by Phytophthora spp., of cocoa (Theobroma cacao) cause combined pod losses of more than 80% in Panama. Biological control of both diseases appeared promising in Peru and is desired by certiWed organic producers in Panama. We evaluated both local and Peruvian fungal antagonists in participatory trials on smallholdings during two complete production cycles. Furthermore, we tested the inXuence of a 3% v/v cane molasses amendment on biocontrol eYcacy, yield and population dynamics of mycoparasites on the cocoa pod. SigniWcant variation was observed between the two years: FPR was more severe in the Wrst year, BP in the second. FPR was signiWcantly reduced by biocontrol agents (BCAs), but not by the molasses amendment. However, BCAs responded diVerently to molasses in both years. All BCAs reduced inoculum production by M. roreri with no consistent eVect of molasses. BCAs had a lesser and more variable eVect on BP, whereas molasses reduced BP slightly by increasing the eYcacy of native antagonists. All BCAs and the molasses amendment enhanced the percentage of healthy pods. Molasses was beneWcial to absolute yield, but only one inoculum improved yield signiWcantly in the Wrst year. Populations of a Peruvian Tricho- derma asperellum isolate remained high for over two months after application to surface-sterilized pods. Molasses had no eVect on estab- lishment or survival of this antagonist or recolonization by any native mycoparasite. The reasons for enhanced biocontrol eYcacy of the molasses formulation requires further investigation. © 2006 Elsevier Inc. All rights reserved.
Keywords: Antagonism; Biological control; Black pod; Clonostachys spp.; Cocoa; Frosty pod rot; Molasses; Moniliophthora roreri; Mycoparasitism; Phytophthora spp.; Theobroma cacao; Trichoderma asperellum
1. Introduction Evans and several Phytophthora spp., predominantly Phy- tophthora palmivora (Butl.) Butl., respectively. In the Bocas del Frosty pod rot (FPR) and black pod (BP) of cocoa (Theo- Toro Province, in the northwest of Panama, both diseases are broma cacao L.) are caused by Moniliophthora roreri (Cif) devastating cocoa production, which is in the hands of mostly indigenous smallholders. Combined pod losses generally exceed 80% under traditional management. This represents * Corresponding author. Current address: CABI-CLARC, Gordon further aggravation from the 35–75% reported by Somarriba Street, Curepe, Trinidad and Tobago. Fax: +1868 663 2859. W E-mail address: [email protected] (U. Krauss). and Beer (1999). Most of the area’s production is certi ed 1 Present address: School of Biological Sciences, University of South- organic, so that only cultural and biological approaches ampton, Bassett, Crescent East, Southampton SO16 7PX, UK. are viable disease management options. Germplasm with
1049-9644/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.biocontrol.2006.06.005 U. Krauss et al. / Biological Control 39 (2006) 232–239 233 resistance to frosty pod, the main disease, is not yet available 2002), but unknown adaptability to Panamanian condi- to growers, although a breeding program with regional trials tions, in addition to some mixed, native inocula. In further exists (Phillips-Mora et al., 2005). discussions, the question as to whether biocontrol eVective- Biocontrol of FPR and BP has shown great promise in ness could be enhanced by the addition of nutrients to the Peru. Krauss and Soberanis (2001, 2002) used native, broad formulation was prioritized. host-range mycoparasites and found mixtures of them par- The main objective of these trials was to evaluate Cen- ticularly eVective, with some yield increases exceeding 15%. tral American and Peruvian biocontrol agents (BCAs) for Similar attempts in Costa Rica yielded variable results (Bat- their eVectiveness against FPR and BP under Panamanian eman et al., 2005; Hidalgo et al., 2003; Krauss et al., 2003). smallholder conditions. Furthermore, we tested the eVect of Very little is known about the eVects of nutrient amend- nutrients in the form of cane molasses on biocontrol ments on the biocontrol eYcacy of fruit rots. Harman et al. eYcacy and antagonist establishment on cocoa pods. (1996) reported increased biocontrol eYcacy of Tricho- derma harzianum against botrytis bunch rot of grape when 2. Materials and methods the antagonist was formulated in 0.5% Pelgel, a mixture of carboxycellulose and gum arabic. Davis et al. (1992) found 2.1. Participatory on-farm Weld trials that Chaetomium globosum formulations containing cellu- lose improved biocontrol eYcacy of pre-harvest apple fruit Participatory Weld trials were conducted in collaboration diseases. Nunes et al. (2001) observed that addition of with the Cooperativa Cacaotera Bocatorreña (COCABO), ammonium molybdate enhanced biocontrol activity of based in Almirante, Bocas del Toro Province, Panama. Cer- Candida sake against post-harvest problems caused by Pen- tiWed organic member smallholder farmers with an interest icillium expansum on apple and pear. Somewhat more in experimentation met with extensionists and researchers research has been done on leaf surfaces, and it may be pos- to discuss disease control options and prioritized realistic sible to draw a comparison, although these play a funda- research objectives at the beginning of the trials. Most mentally diVerent role in plant carbon partitioning: a growers were Ngöbe-Bugle Indians, but descendents of carbohydrate source, as opposed to the carbohydrate sink, Afro-Caribbean and European settlers were also repre- represented by fruit. Even on leaves, the literature is divided sented. (Sutton and Peng, 1993). Populations of desirable yeasts Four farms in the communities Junquito and Rio Este and bacteria can be encouraged eVectively by the applica- Arriba were chosen to install trials in a randomized block tion of carbohydrates, amino acids or mixtures of nutrients design. Relatively large diVerences existed between trial (Andrews, 1992; Kokalis-Burelle et al., 1992). Filamentous sites in terms of cocoa germplasm, farmers’ practice and fungal biocontrol agents (BCAs) may beneWt from nutri- local agroecology. No attempt to standardize these factors ents via enhanced germination, as postulated for Tricho- or deviate from diverse existing practices was made, derma spp. (Hjeljord et al., 2001) and C. globosum (Davis because our aim was to arrive at robust recommendations et al., 1992), or by boosting antibiotic production, as shown of general applicability. Where within-farm variability was for C. globosum (Andrews, 1992). However, mycoparasitic observed for an agroecological feature, treatments were activity tends to be inhibited by nutrients, especially simple installed along the gradient of this feature, so that all treat- sugars (Hjeljord et al., 2001), whereas for example the plant ments experienced similar exposure. Each treatment was pathogen Botrytis cinerea is dependent on exogenous nutri- applied to a row of 20 trees per farm. Trials were carried ents such as sucrose and glucose for germination and pene- out over two complete growing seasons (May 2001–March tration (Guetsky et al., 2002). The eVect of nutrients on 2002 and May 2002–March 2003), with a complete removal competition between pathogen and antagonist, further- of all pods at the beginning of each season. One of the three more, depends on the infection strategy: only necrotrophic agents was replaced by another in the second season, result- pathogens are responsive (Köhl and Fokkema, 1998). ing in an unbalanced factorial arrangement, as reXected in In the present study, we attempted to repeat the Peru- Figs. 1 and 2 and Tables 1 and 2. vian biocontrol success in Panama, initially with native, Native Clonostachys spp. were applied in mixtures of up Central American antagonists, because we believed these to to six isolates per treatment. Mixture MSC contained iso- be best adapted to local agroecological conditions. Only lates AMR07, AMR09, AMR48, APP23, and APP43; these moderate, albeit signiWcant, control levels were obtained in were Clonostachys rosea or Clonostachys byssicola, two spe- early trials on a research station in Costa Rica (Hidalgo cies that can only be distinguished by molecular means et al., 2003) and later in farmers’ Welds (Krauss et al., 2003). (Schroers, 2001). Mixture T2 contained C. byssicola isolates Therefore, collaborators opted to also test a Peruvian iso- AMR37, AMR39, AMR41, AMR42, AMR43, and Clono- late, Trichoderma asperellum Samuels, Lieckfeldt & Niren- stachys cf. byssicola AMR38. Mixture T12 contained C. berg Tr-4,2 of known eVectiveness (Krauss and Soberanis, byssicola AMR55, AMR56, AMR57 and again Clonosta- chys spp. APP23 and APP43. Inoculum was produced on 2 The International Mycological Institute originally identiWed this iso- Guata (Krauss et al., 2002). Only the Peruvian T. asperel- late as Trichoderma longibrachiatum (IMI 382482). However, subsequently lum Tr-4 was applied as single strain. This fungus was pro- Gary Samuels (USDA) reclassiWed it as T. asperellum. duced on rice (Krauss et al., 2002). Both production 234 U. Krauss et al. / Biological Control 39 (2006) 232–239
Healthy 2001-2002 Frosty Pod Rot Black Pod 100% hi 80% f defg h fg h fgh ef i a h efg cde gh 60% gh cde
40%
20% a b a a def def a bcd 0% bc Water Water Water Water Molasses Molasses Molasses Molasses Control Mixture Mixture Mixture MSC T2 T12
2002-2003 100% cdef 80%
60% b de fg bcd i bc bcd fg ef ab bc ab i a i
40%
20%
0% h def ef ef gh fg cde def Water Water Water Water Molasses Molasses Molasses Molasses Control Mixture Mixture Trichoderma T2 T12 asperellum Tr-4
Fig. 1. Percentage of diseased and healthy cocoa pod in biocontrol trials conducted in Panama over two growing seasons. Bar segments carrying the same letter do not diVer at P D 0.05 (comparison within category, across years).
40 30
% 20 na 2002/03
Sporulation 10 na 2001/02 0 Control Mixture Mixture Mixture Trichoderma MSC T2 T12 asperellum Tr-4
Fig. 2. Percentage of FPR-infected pods reaching sporulation stage in biocontrol trials in Bocas del Toro, Panama in two growing seasons (na, not appli- cable; treatment not applied). methods yielded over 107 cfu ml¡1 after extraction for the onist suspension was applied either in water or with 3% v/v respective fungi. Extraction was done in the Weld, based on cane molasses (Ingenio Atirro, Turrialba, Costa Rica) at a the previous calibration of each inoculum batch in the lab- rate of approximately 3 £ 1012 cfu ha¡1, using pressurized oratory, adjusting inoculum to a total of 106 cfu ml¡1 with (3–4 kg cm¡2) knapsack sprayers (Protecno 17, Productos equal proportions of all antagonists in mixtures. The antag- Tecnológicos, San Salvador, El Salvador). An absolute con- U. Krauss et al. / Biological Control 39 (2006) 232–239 235
Table 1 Rica’s Atlantic lowlands. Cocoa pods, approximately two Number of frosty pod rot-infected pods (per 20 trees) reaching the sporu- months before maturity, were surface-sterilized by wiping lation stage in four biocontrol plots with or without cane molasses (3% v/ them with 95% alcohol and subsequently spray-inoculated v) in the formulation, in Bocas del Toro, Panama, observed over two 6 ¡1 growing seasons with T. asperellum Tr-4 (concentration: 10 cfu ml ) or the control treatment to incipient run-oV using a hand-held, Season Treatment Molasses pressurized sprayer (2 l sprayer, Volpi & Bottoli, Piadena, No Yes Italy). The trees were spaced 2 m £ 4 m with heights of 3– 2001–2002 Control 156.5f 88.9d 4 m. Thirty pods per treatment were chosen randomly d e Mixture MSC 86.7 106.0 across clones on a total of 7–10 trees. The experiment com- Mixture T2 35.2a 68.2c Mixture T12 71.4c 43.5ab pared four treatments with a 2 £ 2 factorial arrangement: Trichoderma asperellum Tr-4 na na pods inoculated or not with T. asperellum Tr-4 and water 2002–2003 Control 64.2c 114.4e versus molasses solution (3% v/v) as a carrier. Mixture MSC na na Samples were taken in weekly intervals over a period of Mixture T2 87.7d 76.6cd 10 weeks. Three peel discs (diameter 12 mm) were cored Mixture T12 51.0b 45.1b from each of seven replicate pods per treatment and date; ab ab Trichoderma asperellum Tr-4 42.9 41.3 the remainder of the treated pods served as replacement for na, not applicable; treatment not administered. pods lost due to rots, especially after injury. The surface a,b,c,d,e,f V Values followed by the same letter do not di er at P D 0.05. layer of the discs was removed with a scalpel and air-dried (ca. 3 days) in the laboratory. The dried discs were then Table 2 placed, with their peel facing downwards, onto agar plates Number of healthy pods (per 20 trees) harvested in four biocontrol plots fully pre-colonised with P. palmivora as a bait host (Piper with or without cane molasses (3% v/v) in the formulation, in Bocas del Toro, Panama, observed over two growing seasons et al., 2000) that is highly susceptible to mycoparasitism (Ten Hoopen et al., 2003). The development of T. asperel- Season Treatment Molasses lum Tr-4 and/or native mycoparasites on the host was No Yes observed for approximately one week, scored as presence/ 2001–2002 Control 90.1cde 109.2gh absence and converted into colonisation percentages on 21 efgh i Mixture MSC 104.8 133.4 peel disks per treatment and date. Mixture T2 90.6cdef 104.2efgh Mixture T12 108.9gh 100.1defg Survival of applied Trichoderma and re-colonisation Trichoderma asperellum Tr-4 na na dynamics of other epiphytic mycoparasites were graphed. No simple curve seemed to Wt the time-course, and treat- 2002–2003 Control 87.5bcd 118.8hi Mixture MSC na na ment means were compared using the appropriate general Mixture T2 74.2ab 70.3a linear model on Genstat 5, with repeated measurements in Mixture T12 79.1abc 116.5h time computed as recommended by Zar (1996). Trichoderma asperellum Tr-4 99.0defg 105.0fgh na, not applicable; treatment not administered. 3. Results a,b,c,d,e,f,g,h,i Values followed by the same letter do not diVer at P D 0.05. 3.1. Frosty pod rot (FPR) trol and a 3% molasses solution served as controls. A total of eight, monthly applications were administered per sea- Overall, more FPR was observed in 2001/2002 (51.6%) son (May–March), starting with the Xowering peak in May, than in 2002/2003 (42.2%) (P 6 0.001). The control agent with the last application in December. (P < 0.05), but not molasses (P D 0.691) had a signiWcant Phytosanitary pod removal was applied to all treatments in eVect on FPR incidence. However, the agent interacted sig- fortnightly intervals. At the same time, the following trial data niWcantly with year and year £ molasses (both P < 0.001). were recorded: healthy, mature pods and all diseased pods, The interaction agent £ molasses narrowly failed to reach independent of their age, were removed and quantiWed. FPR- signiWcance (2 D 9.41, df D 4). This pattern of interaction infected pods were further distinguished into sporulating ones means that the overriding factor for biocontrol of FPR was and those exhibiting earlier symptoms. Percentage data and the variability encountered in diVerent years and that this healthy pod counts were analyzed using the appropriate gen- also inXuenced the reaction of BCAs to molasses, albeit to a eral linear model for binomial or Poisson-distributed data on lesser extent. Genstat 5 (Genstat 5 Committee of the Statistics Department Molasses application alone reduced FPR compared with of Rothamsted Experimental Station, 1993), respectively. the absolute control in the Wrst year, but increased FPR in the second year (Fig. 1). In the Wrst year, all agents reduced 2.2. EVect of cane molasses on establishment and survival of FPR in the absence of molasses, but only the mixed inocu- Trichoderma asperellum on cocoa pods lum T12 did so in the presence of molasses. The mixture MSC was found to be the least eVective and was replaced This Weld survival trial was conducted in the clonal gar- by the single-strain inoculum T. asperellum Tr-4 in the den of CATIE’s La Lola Experimental Station in Costa following year. Contrary, in the second year, none of the 236 U. Krauss et al. / Biological Control 39 (2006) 232–239 treatments reduced the disease in the absence of molasses, was due to the beneWcial eVect of molasses on T2 during the but T12 and Tr-4 did so in the presence of molasses. Wrst year. In this year, and only in the presence of molasses, For the percentage of FPR-infected pods that reached T2 and MSC reduced BP incidence compared with the con- sporulation, agent was signiWcant (P 6 0.002), while molas- trols. In the second year, in which MSC was replaced by Tr- ses had no eVect (P D 0.731) and did not interact with any 4, two treatments, T2 and Tr-4, reduced BP, but this time other factor (2 6 8.14, df D 4). The two seasons diVered sig- only in the absence of molasses (Fig. 1). niWcantly (P < 0.001), but agent and year interacted signiW- cantly (P < 0.01), probably mostly due to the unbalanced 3.3. Healthy pod yield design rather than variable behavior of BCAs (Fig. 2). All BCAs, except MSC, reduced the percentage of and, thus, The percentage healthy pods (Fig. 1) is a function of disseminated pathogen inoculum. T12 was most eVective, losses due to FPR and BP, two diseases that showed con- followed by T2 and Tr-4 (Fig. 2). In the second year, a trasting trends. All factors and their Wrst-order interactions, higher percentage (29.8%) sporulated than in the Wrst year except molasses £ year, were signiWcant. All BCAs (24.9%). (P 6 0.021) and molasses in the formulation (P <0.001) The total number of sporulating pods (Table 1) is a func- increased the percentage of healthy pods. A higher percent- tion of disease incidence (Fig. 1) and sporulation incidence age of healthy pods was recorded in the second trial year (Fig. 2), and is directly related to inoculum to be produced than in the Wrst one (P < 0.001). However, the eVect of bio- by M. roreri. All BCAs reduced the number of sporulating control was more pronounced in 2001/2002, when all agents pods signiWcantly. Agent (P < 0.001) and year (P <0.001) led to a signiWcant increase in the percentage of healthy were signiWcant. Molasses had no simple eVect but inter- pods in the presence and absence of molasses. In 2002/2003, acted with agent (P < 0.01) and agent £ year (P <0.001). only Tr-4 achieved this, while T12 did so in the presence of The interaction agent £ year was also signiWcant molasses only. The addition of molasses increased the per- (P < 0.001). Again, T12 was most eVective, followed by T2, centage of healthy pods for T12 and the control consis- Tr-4 and Wnally MSC. As for FPR incidence, more pods tently in both years (Fig. 1). sporulated in 2001/2002 than in 2002/2003 but, contrary to Absolute yield was measured as number of healthy pods disease incidence, the BCAs exhibited a clearly diVerential harvested (Table 2). This measure is more variable, and reaction towards molasses in the two seasons. BCAs did not signiWcantly increase absolute yield in the In 2001/2002, all agents reduced the number of sporulat- second year, although they increased the percentage ing pods in the absence of molasses (Table 1). Only MSC healthy pods (Fig. 1). In the second year, T2 signiWcantly failed to do so in the presence of molasses. In 2002/2003, in decreased the number of healthy pods in the presence of which MSC was not applied, all agents reduced the number molasses. Overall, the molasses formulation was found to of sporulating pods in the presence of molasses, but T2 was be beneWcial for yield (P < 0.001). This beneWcial eVect was the only treatment that failed to do so in the absence of clear for the control (both years), for MSC (year 1), and for molasses, whereas this had been the most eVective treat- T12 (year 2). None of the interactions were signiWcant ment in year one. While molasses application alone reduced (2 6 5.85). the number of FPR-infected pods that reached sporulation compared with the absolute control in the Wrst year, it 3.4. EVect of cane molasses on establishment of Trichoderma increased their number in the second year. In 2001/2002, asperellum on cocoa pods and recolonization by native molasses improved the anti-sporulation eVect of T12 but mycoparasites seriously compromised the eVectiveness of T2. In contrast, in 2002/2003, molasses had no signiWcant eVect on The surface sterilization prior to application and evalua- any BCA with respect to numbers of sporulating pods tion did not completely eliminate all cocoa pod-colonizing (Table 1). fungi, some of which are known for their subcuticular or even endophytic growth habit (Ten Hoopen et al., 2003 and 3.2. Black pod (BP) references therein). Application of T. asperellum Tr-4 to surface-sterilized cocoa pods signiWcantly (P <0.001) The incidence of BP behaved almost reverse to FPR inci- increased the populations of Trichoderma spp. High popu- dence. In 2001/2002, less BP (31.2%) was observed than in lation levels (>50%) were maintained for at least Wve weeks 2002/2003 (37.5%) (P < 0.001). Whereas the molasses-only (Fig. 3). The addition of 3% molasses to the formulation application increased BP incidence compared with the had no eVect (P D 0.445). absolute control in the Wrst year, it reduced BP in the fol- Other competing fungal genera were largely unaVected lowing year (P < 0.05). Overall, only MSC reduced BP, by the application of T. asperellum Tr-4: Clonostachys spp. whereas T12, the most eVective BCA against FPR, (P D 0.774), Fusarium spp. (P D 0.538), Gliomastrix muro- increased it (Fig. 1). rum (P D 0.052) and pooled others (P D 0.131). The only Molasses slightly but signiWcantly (P D 0.005) reduced exceptions were pooled Aspergillus and Penicillium spp., BP incidence from 35.0% to 33.5%, but also interacted with which were signiWcantly (P < 0.001) more abundant on con- agent £ year (P < 0.01). The improvement of disease control trol pods than on Trichoderma-treated pods. None of these U. Krauss et al. / Biological Control 39 (2006) 232–239 237
Trichoderma Clonostachys
100.0 100
80.0 80
60.0 60
40.0 40
20.0 20 Pod surface colonisation (%)
0.0 Pod surface colonisation (%) 0 0123495 67108 0123495 67108 Weeks afterinoculation Weeks after inoculation
Fusarium Gliomastrix murorum Tr-4 in water 100 100 Tr-4 in molasses 80 80 Water control 60 60 Molasses control 40 40 20 20 0 0 Pod surface colonisation (%) Pod surface colonisation (%) 0123495 67108 0123495 67108 Weeks after inoculation Weeks after inoculation
Aspergillus and Penicillium Other Genera 100 100
80 80
60 60
40 40
20 20
Pod surface colonisation (%) 0
Pod surface colonisation (%) 0 0123495 67108 0123495 67108 Weeks after inoculation Weeks after inoculation
Fig. 3. Percentage pod colonization with common mycoparasites over time after application of Trichoderma asperellum Tr-4, with or without cane molas- ses (3% v/v), to surface-sterilized cocoa pods. fungi were inXuenced by the presence of molasses 4. Discussion (0.107 6 P 6 0.887). The population dynamics over time are shown in Fig. 3. We observed reduced disease losses and increased yield Clonostachys populations appeared reduced after surface in the molasses-only control, compared with the absolute sterilization of the pods (week 0), at least in the control, but control. This suggests that molasses either enhanced natu- stabilized at over 90% pod surface colonization after one rally occurring BCA populations or acted as a foliar/fruit week, irrespective of treatment. Fusarium spp. occurred fertilizer, conveying improved resistance and increasing infrequently and exhibited no clear trend in time. G. muro- yield. The later seems less likely, because percentage yield rum, competitive saprophyte rather than a mycoparasite, improvement (36.1% and 19.7% in 2001/2002 and 2002/ was an early pod colonizer, which peaked in week two; later 2003, respectively) was greater than absolute yield improve- (week four onwards) it was no longer detected. The oppo- ment (21.2% and 13.3% in 2001/2002 and 2002/2003, respec- site tendency was observed for Aspergillus and Penicillium tively), i.e. molasses did not increase the net number of pods spp.: their populations were very low during the Wrst month formed. Enhancement of natural antagonists, especially after installation of the experiment. After four weeks on bacteria and yeasts, has been reported on leaves of cereals uninoculated pods and seven weeks on Trichoderma-inocu- and sycamore (Andrews, 1992 and references therein). Pop- lated pods, population had grown substantially. Other ulation shifts and increases of overall populations of chitin- pooled genera Xuctuated strongly with a slight increase olytic members of naturally occurring epiphytes, associated over time (Fig. 3). with the applications of a chitin formulation, decreased the 238 U. Krauss et al. / Biological Control 39 (2006) 232–239 number of leafspots on leaves of peanut (Kokalis-Burelle gistasis (Hjeljord and Tronsmo, 1998). It should also be et al., 1992). Guetsky et al. (2002) found that, in the pres- noted that inoculum of T. asperellum Tr-4 was extracted ence of sucrose (0.1% w/v), B. cinerea germination on from its rice substrate in the Weld, immediately prior to detached strawberry leaves was reduced from 56.7% to application, which may have provided some exogenous 30.7% by the addition of leucine. However, Hjeljord et al. nutrients in the form of rice particles in the application mix- (2000) reported increased strawberry rot (Botrytis cinerea ture, whereas Clonostachys spp. were extracted from the and Mucor piriformis) on plants sprayed with sucrose (1% inert Guata (Krauss et al., 2002). Fresh Trichoderma w/v) compared with the water control. We found no evi- conidia are less dependent on exogenous nutrients than dence of antagonistic Wlamentous fungi beneWting from older ones. However, diVerent species of Trichoderma cane molasses amendment. Bacteria and yeasts merit a respond diVerently to diVerent types of nutrients (Nelson closer look. The survival of some antagonistic bacteria on et al., 1988). No study so far has covered T. asperellum. In banana (Musa AAA) leaves was enhanced by cane molas- better investigated soil systems, Trichoderma spp. are rela- ses and other nutrient amendments in greenhouse assays tively sensitive to fungistasis compared with other soil (Ruiz-Silvera et al., 1997a). However, under Weld condi- fungi, but not extremely so. Fungistatic sensitivity is nega- tions, cane molasses alone increased Black Sigatoka tively correlated to spore size (Steiner and Lockwood, (Mycosphaerella Wjiensis) infection and, as an amendment 1969). Amongst Trichoderma spp., the conidia of T. asperel- with bacterial BCAs, did not inXuence biocontrol eYcacy lum are in the average range, with dimensions of (2.8¡)3.4– (Ruiz-Silvera et al., 1997b), while biocontrol eVected by 3.6 (¡7.0) £ (2.4¡) 3–4 (¡6) m (Samuels et al., undated). yeasts was enhanced by complex nutrient amendments Various other Trichoderma spp. coped with nutrient stress (Arango, 2000). On detached strawberry leaves, Guetsky better under acidic conditions (pH 4.3) than neutral ones et al. (2002) found that the eVects of nutrient amendments (Danielson and Davey, 1973), but such in vitro simulations on biocontrol eYcacy of a mixture of Pichia guilermondii are not necessarily representative of germination on natural and Bacillus mycoides were erratic. DiVerent supplements substrates (Nelson et al., 1988). How fungistasis may aVect had diVerent eVects with some nutritional supplements the eYcacy of the applied BCAs, and speciWcally T. asperel- almost nullifying the eVects of the control agents and some lum Tr-4, remains to be studied in more detail. enhancing it greatly. Bateman et al. (2005) speculated that residual cane Molasses is a complex nutritional supplement which molasses from the fermentation process to mass-produce might contribute to the inconsistent eVects on biocontrol. Clonostachys spp. inoculum (Krauss et al., 2002) may have The total carbohydrate content of the molasses was deter- negatively aVected the competition between M. roreri and mined as being 59.1% w/w by the laboratory of the Univers- its antagonists in trials in Costa Rica. In our trials in Pan- idad de Costa Rica, using the Lane–Eynon method. This is ama, the necrotrophic pathogen P. palmivora showed no lower than the average 72.5% w/w commonly found in cane clearer response to BCAs formulated in molasses than M. molasses (Oregon State University, undated). Typically, roreri, a biotroph during the early stages of its infection cane molasses has a higher carbohydrate content than beet cycle (Evans et al., 2002). Instead, an opposite trend for molasses and a ratio of C12 sugars to other sugars of 1.7, as these two pathogens was noted. This applied to FPR inci- compared with a ratio of 105 for beet molasses. Cane dence, presumably dominated by the biotrophic stage, as molasses contains approximately 7.5% w/w nitrogenous well as to sporulation of infected pods, presumably domi- compounds, beet molasses twice that much (15.6% w/w). nated by the saprophytic stage of M. roreri. Thus, in our Other compounds are in a similar range (Oregon State Uni- pathosystems, we could not reproduce the observation of versity, undated). Köhl and Fokkema (1998) that necrotrophic pathogens Cane molasses amendment had no consistent eVect on beneWt more from nutrient amendments than biotrophs. biocontrol eYcacy. Instead, the various treatments Furthermore, none of the facultative mycoparasites and responded diVerently in the two trial seasons. Nutrient other saprophytic pod fungi responded measurably to deposits available to microbes on leaves are highly variable molasses amendments during recolonization. and depend on a multitude of factors (Andrews, 1992). Only T. asperellum Tr-4 reduced FPR and BP simulta- These merit investigation, especially on fruit, as informa- neously. Although only data for one year are available for tion there is extremely scarce, although fruit are the com- this BCA, these are promising. This Peruvian isolate, mercial product of the majority of crops. While T. applied in water, was also the best treatment in participa- asperellum Tr-4 appeared indiVerent to molasses both, in tory on-farm trials in neighboring Costa Rica, where a 34% biocontrol and Weld survival trials, a generalization for yield increase was recorded in 2001/2002, while the local other fungi is not possible. Some agents responded posi- mixture T2 improved yield by 21% (Krauss et al., 2003). tively, and yield, on the whole, beneWted from the molasses For comparison, Krauss and Soberanis (2002), in Peru, amendment. harvested an additional 19.7% of healthy pods with T. Fungistasis is the inhibition of germination and growth asperellum Tr-4 applications. This indicates that T. asperel- of fungal cfus caused by nutrient stress. In addition to lum Tr-4 adapted well to Central American conditions. On conidia, the BCA extraction procedures yielded some the other hand, Bateman et al. (2005) observed no eVect hyphal fragments, which tend to be less susceptible to fun- on yield by either T. asperellum Tr-4 or a mixture of three U. Krauss et al. / Biological Control 39 (2006) 232–239 239
Peruvian C. byssicola strains with T. asperellum Tr-4 in a ucts to control Botrytis cinerea and Mucor piriformis in greenhouse Costa Rican experimental station. Unfortunately, the strawberries. Biol. Control 19, 149–160. authors did not analyze disease incidence data. The large Hjeljord, L.G., Stensvand, A., Tronsmo, A., 2001. Antagonism of nutrient- activated conidia of Trichoderma harzianum (atroviride) P1 against variability both, across seasons and within a relatively Botrytis cinerea. Phytopathology 91, 1172–1180. small geographic area, suggests that important ecological Köhl, J., Fokkema, N.J., 1998. Strategies for biological control of necro- factors inXuencing the pathogen–antagonist interaction on trophic fungal foliar pathogens. In: Boland, G.J., Kaykendale, L.D. the cocoa pod are yet to be elucidated before we can (Eds.), Plant–Microbe Interactions and Biological Control. Marcel improve biocontrol eYcacy in an informed and targeted Dekker, New York, USA, pp. 49–88. Kokalis-Burelle, N., Backman, P.A., Rodríguez-Kábana, R., Ploper, L.D., manner. 1992. Potential for biological control of early leafspot of peanut using Bacillus cereus and chitin as foliar amendments. Biol. Control 2, 321–328. Acknowledgments Krauss, U., Soberanis, W., 2001. Biocontrol of cocoa pod diseases with mycoparasite mixtures. Biol. Control 22, 149–158. V This work was funded by USDA-ARS, DGIS, Cocoa Krauss, U., Soberanis, W., 2002. E ect of fertilization and biocontrol application frequency on cocoa pod diseases. Biol. Control 24, 82–89. Research UK, and the OLIN Foundation, and was man- Krauss, U., Martínez, A., Hidalgo, E., Ten Hoopen, M., Arroyo, C., 2002. aged by CABI Bioscience, CATIE, and COCABO. We are Two-step liquid/solid state scaled-up production of Clonostachys rosea. most grateful to all participating farmers and to Robert Mycol. Res. 106, 1449–1454. Mack for their enthusiasm and thought-provoking discus- Krauss, U., Ten Hoopen, M., Hidalgo, E., Martínez, A., Arroyo, C., Gar- sions. We also thank the University of Bath for support cía, J., Portuguez, A., Sánchez, V., 2003. Manejo Integrado de la monili- asis (Moniliophthora roreri) del cacao (Theobroma cacao) en through their student placement program. Maribel Mora, Talamanca, Costa Rica. Agroforestería en las Américas 10, 52–58. Armando Portuguez and Miguel Sanabria provided techni- Nelson, E.B., Harman, G.E., Nash, G.T., 1988. Enhancement of Tricho- cal assistance. derma-induced biological control of pythium seed rot and pre-emer- gence damping-oV of peas. Soil Biol. Biochem. 20, 145–150. Nunes, C., Usall, J., Teixidó, N., Miró, M., Viñas, I., 2001. Nutritional References enhancement of biocontrol activity of Candida sake (CPA-1) against Pen- icillium expansum on apples and pears. Eur. J. Plant Pathol. 107, 543–551. Andrews, J.H., 1992. Biological control in the phyllosphere. Annu. Rev. Oregon State University, undated. Molasses