Evaluation of mycoparasites as biocontrol agents of Rosellinia root rot in cocoa

Biological Control 27 (2003) 210–227 www.elsevier.com/locate/ybcon

Evaluation of mycoparasites as biocontrol agents of Rosellinia root rot in cocoa Ramon A. Mendoza Garc
ıa,a G. Martijn ten Hoopen,b Donald C.J. Kass,c Vera A. S
anchez Garita,d and Ulrike Krausse,* a

CATIE/MIP/AF/NORAD, Apdo. Postal P-116, Managua, Nicaragua b DGIS-CABI Bioscience, c/o CATIE, 7170 Turrialba, Costa Rica c 11 Riverside Drive, Apt. 4PE, New York, NY 10023, USA d CATIE, 7170 Turrialba, Costa Rica e CABI Bioscience, c/o CATIE 7170, Turrialba, Costa Rica Received 6 August 2002; accepted 26 December 2002

Abstract Rosellinia spp. cause substantial mortality of cocoa (Theobroma cacao), especially in acidic soils rich in organic matter (O.M.) and, reportedly, low available phosphate. Cultural and chemical control are relatively ineffective and a supplementary biocontrol strategy would be highly desirable. Our aim was the isolation and evaluation of promising mycoparasites against Rosellinia of cocoa. We isolated 45 mycoparasites using a baiting technique. Three of them and a commercial isolate were prioritized for further evaluation based on their broad hostrange and/or effectiveness in pot bioassays. Because Rosellinia is favored by the same soil factors as mycoparasites, we investigated the effect of soil pH (range 4–6), O.M. (6–12%), and phosphate (0–1000 mg kg
1 ) on their interaction. In vitro, both pathogens and antagonists grew well on a wide range of phosphate (0–9500 mg kg
1 ) and at various pH (3.0–10.0) levels. The pH optimum usually coincided with the soil of origin and the phosphate optimum depended on the phosphate source used. In pot bioassays using natural soils, mixtures of antagonists exhibited more control than single strains and mixtures were effective over a wider range of soil conditions. High O.M. favored the pathogen more than its biocontrol agents. Soil pH interacted with the treatments such that biocontrol was most efficient at higher pH values and with the highest mixtures of antagonists. The addition of phosphate enhanced plant growth but had little effect on biocontrol efficacy. We conclude that mixtures of mycoparasites should be field tested in combination with liming and removal of O.M. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Antagonism; Biological control; Clonostachys spp.; Dematophora necatrix; Cocoa; Mycoparasitism; Organic matter; Phosphate; Potato; Root rot; Rosellinia spp.; Solanum tuberosum; Soilborne pathogen; Soil pH; Theobroma cacao; Trichoderma spp.

1. Introduction Black root rot or Rosellinia root rot in cocoa (Theobroma cacao L.) is an important disease in Latin America and the Caribbean (Aranzazu et al., 1999; Cadavid, 1995; Merchan, 1989; Waterston, 1941). According to Aranzazu et al. (1999), over 50% of the cocoa producing farms in some areas of Colombia are affected by Rosellinia. In one estate, between 0.3 and 4.4% of cocoa trees were lost to Rosellinia spp. within two years.

* Corresponding author. Fax: +556-0606. E-mail address: [email protected] (U. Krauss).

Seven years after planting, less than half of the original population had survived in those disease foci (Cadavid, 1995). In the Bigu
a-Peru
ıbe region, S~ao Paulo, Brasil, up to 20% of three-year-old cocoa trees died (Feitosa and Pimentel, 1991). In CATIEÕs cocoa germplasm bank in Cabiria (Costa Rica), 45 clones, some of them unique, were lost to a complex of Ceratocystis fimbriata Ellis and Halsted and Rosellinia between 1997 and 2001, which is equivalent to 1.4% per year. The causal agents of this soilborne disease in cocoa are Rosellinia pepo Pat. and Rosellinia bunodes (Berk. & Br.) Sacc. A third species, Rosellinia paraguayensis Starb., has only been recorded on cocoa in Grenada (Waterston, 1941). R. pepo and R. bunodes are known

1049-9644/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1049-9644(03)00014-8

R.A. Mendoza Garc
ıa et al. / Biological Control 27 (2003) 210–227

throughout the world, especially in tropical America, the West Indies, West Africa, and Southern Asia. They cause root rot in a large and diverse number of economically important crops like banana, cocoa, coffee, tea, citrus, rubber, avocado, and various important timber species (Aranzazu, 1996; Booth and Holliday, 1972; Saccas, 1956; Sivanesan and Holliday, 1972; Waterston, 1941). In coffee in Colombia, losses of $489:00 ha
1 yr
1 due to Rosellinia have been reported (Bautista and Magdiel, 2000). Although Rosellinia is considered a facultative and weak pathogen (Aranzazu, 1996; Waterston, 1941), control of the disease is difficult once it erupts. Woody perennial crops add further challenges, especially with respect to delivery of the control agent to the target zone. Cultural, chemical, and biological control measures have been investigated as to their effectiveness in controlling Rosellinia root rots with varying results. As early as in 1908, Stockdale (1908) described cultural methods to control the disease. Nowell (1916) expanded these methods and to date they are one of the few measures available to farmers to manage the disease. Cultural control measures consist generally of three steps. The first step is to isolate the diseased tree(s)/area(s), using trenches and/or root pruning (Aranzazu, 1997; Aranzazu et al., 1999; Nowell, 1916). The second step consists of the removal and burning of coarse woody debris (CWD) in the form of the infected trees, including the infected root system and CWD lying on the ground (Aranzazu et al., 1999; Nowell, 1916; Waterston, 1941). The third and final steps are the application of lime to increase the pH and facilitate the decomposition of organic material (Aranzazu et al., 1999; Nowell, 1916). Solarization of diseased areas might also help in eradicating the pathogen (Aranzazu et al., 1999; Nowell, 1916; Sztejnberg et al., 1987). The use of fungicides to control Rosellinia has proven relatively ineffective and uneconomical (Aranzazu et al., 1999; Bautista and Rivera, 1997; Cubillos, 1988) and has even been reported to accelerate disease development by killing the native, antagonistic microflora (Aranzazu et al., 1999). In coffee, only one fungicide out of five tested, tolclofos methyl (Rizolex), was successful in controlling Rosellinia over a three-year period but only when applied at a dose of 72 kg ha
1 (15 g per planting hole, 4800 plants ha
1 ) twice a year. The morpholine fungicide tridemorph showed no effect in controlling Rosellinia root rot in cocoa (Cubillos, 1988). Since numerous fungicides were inhibitory in vitro (Achicanoy, 1989), delivery to the target zone is likely to have been the limiting factor in perennial crops. Due to the high costs of the fungicides, their relative ineffectiveness and the damage to the environment, alternative methods to control Rosellinia in cocoa are necessary. In the last few decades, microbial control of soilborne pathogens has received much attention and

211

numerous reviews have been published (e.g., Deacon, 1991; Harman, 2000; Papavizas, 1973). Several studies have been done on biological control of Rosellinia spp. (Castro, 1995; Esquivel et al., 1992; Freeman et al., 1986; Ruiz and Leguizam
on, 1996; Sztejnberg et al., 1987) but no work has been published on biocontrol in cocoa. Shirata et al. (2000) and Yoshida et al. (2001) focused on using bacterial metabolites derived from Janthinobacterium lividum and Bacillus amyloliquefaciens, respectively. Although inhibitory to Rosellinia necatrix Prill. (anamorph: Dematophora necatrix Hartig) in vitro, they are likely to face the same delivery problems as fungicides under field conditions. Most work on biocontrol of Rosellinia concentrated on mycoparasites of the genus Trichoderma. Freeman et al. (1986) and Sztejnberg et al. (1987) tested Trichoderma hamatum (Bon.) Bainier and Trichoderma harzianum Rifai against R. necatrix, while Esquivel et al. (1992), Castro (1995), and Ruiz and Leguizam
on (1996) evaluated Trichoderma koningii Oudemans as biocontrol agents (BCAs) of R. bunodes strains that attacked coffee. While results from in vitro or greenhouse experiments were very promising with Trichoderma spp. reducing disease incidence by up to 85% (Esquivel et al., 1992), the only two field experiments in which Trichoderma spp. were tested against R. necatrix showed no disease control (Sztejnberg et al., 1987; Kojima, 1987, cited by Matsumoto, 1998). In Colombia, where Rosellinia root rot is particularly serious, the disease is mostly associated with fairly acid soils rich in organic matter (O.M.) with a high soil moisture level (L
opez and Fern
andez, 1966), but Waterston (1941) and Cadavid (1995) did not find any relation between acidity and disease occurrence in Colombia and the West Indies, respectively. Furthermore, Waterston (1941), and L
opez and Fern
andez (1966) suggested that disease development is favored by soils with low phosphate contents, although no supporting experimental data were presented. Trichoderma spp. and Gliocladium/Clonostachys (Schroers et al., 1999) are acidophilic soil inhabitants which thrive on O.M. and wet conditions (Papavizas, 1985). Thus, these potential BCAs benefit from the same conditions that favor the pathogen. To date, the relative importance of these factors on the equilibrium between pathogen and BCAs has not been investigated. The objective of this study was to isolate mycoparasites against Rosellinia and identify candidates that could be developed into effective biocontrol agents. This included an evaluation of antagonism by the mycoparasites as single strains or in mixtures towards Rosellinia. Furthermore, we investigated the effect of soil pH, O.M., and phosphate content on the outcome of the antagonistic interaction in the greenhouse using natural soils.

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2. Materials and methods 2.1. Fungal cultures Three isolates of Rosellinia, RB1, RB3, and RB4, were obtained from roots of naturally infected cocoa, located in the germplasm bank of CATIE in Cabiria. One isolate, RB2, was isolated from potato roots (Solanum tuberosum L.) collected in Tierra Blanca de Cartago, Costa Rica. Stock cultures were stored on acidified potato dextrose agar (PDAA, pH adjusted to 3.5 using 25% v/v lactic acid). Mycoparasites of Rosellinia were collected twice in order to obtain a large and versatile selection of possible biocontrol candidates, once at the end of the dry season (March/April 2000) and once during the rainy season (July/September 2000). In disease foci in the germplasm bank of CATIE and affected fields in Tierra Blanca, soil samples were collected from the rhizosphere near apparently healthy plants, about 30 cm away from the stem of the plant and at a depth of 30 cm. Soil samples were then brought to the laboratory, thoroughly mixed and subsequently five sub-samples were taken. These were left to air-dry at ambient temperature (23–27 °C) to curb bacterial growth. Mycoparasites were isolated using the

precolonized plate method as described by Foley and Deacon (1985) using RB1 and RB2 as hosts for soils from CATIE and Tierra Blanca, respectively. Mycoparasites found growing over the Rosellinia mycelium where isolated, purified, and stored on potato dextrose agar (PDA, Merck KGaA, Darmstadt, Germany) (Table 1). Two commercially available mycoparasites, T. harzianum strain T-22 (isolated from Root Shield, Bio Works, Geneva, NY, USA) and Trichoderma virens (Miller et al.) Arx strain GL-21 (isolated from Soil Guard 12G, Thermo Trilogy, USA), which are widely reported as highly effective antagonists of soilborne pathogens (Harman, 2000; Lumsden et al., 1996), were added for comparison. For production of Rosellinia (RB1) inoculum, glass bottles (capacity 1 L) were filled with 100 g wheat seeds and 100 ml water and autoclaved for 35 min on two consecutive days. The flasks were inoculated with 4–6 fungal disks of two-week-old cultures of the Rosellinia isolate. The bottles were incubated in constant darkness at ambient temperature (23–27 °C) until the wheat seeds were completely covered by mycelium, which took approximately 3–4 weeks. Spore suspensions of the mycoparasites were obtained by flooding PDA plates with the respective an-

Table 1 Antagonists used in this study, their isolation host, isolation date, and origin (all from Costa Rica and soil, except T-22 and GL-21) Species

Accession No.

Isolation host

Isolation date

Location

Trichoderma sp. Trichoderma sp. Trichoderma sp. Trichoderma harzianum Fusarium sp. Fusarium sp. Fusarium sp. Fusarium sp. Penicillium sp. Unidentified Clonostachys rhizophaga Clonostachys sp. Trichoderma sp. Trichoderma sp. Trichoderma sp. Trichoderma sp. Trichoderma sp. Clonostachys sp. Trichoderma sp. Unidentified Trichoderma sp. Trichoderma sp. Trichoderma sp. Clonostachys sp. Clonostachys sp. Clonostachys cf. byssicola. Clonostachys sp. Trichoderma virens Trichoderma harzianum

ARB1 ARB2 ARB3 ARB4 ARB5 ARB6 ARB7 ARB8 ARB9 ARB10 ARB11 ARB12 ARB13 ARB15 ARB16 ARB17 ARB18 ARB19 ARB20 ARB21 ARB22 ARB33 ARB34 ARB35 ARB36 ARB37 GC GL-21 T-22

RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB2 RB2 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB1 RB2 n.a.a n.a.

March, 2000 March, 2000 March, 2000 March, 2000 March, 2000 March, 2000 April, 2000 March, 2000 March, 2000 April, 2000 March, 2000 March, 2000 July, 2000 July, 2000 July, 2000 July, 2000 July, 2000 July, 2000 July, 2000 July, 2000 July, 2000 September, 2000 September, 2000 September, 2000 September, 2000 September, 2000 n.k.b n.k. n.k.

Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Tierra Blanca Tierra Blanca Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Cabiria Tierra Blanca USA USA

a b

n.a., not applicable. n.k., not known.

R.A. Mendoza Garc
ıa et al. / Biological Control 27 (2003) 210–227

tagonist with sterile distilled water (SDW). Spore concentrations were determined using a hemacytometer and adjusted to the desired levels with SDW. 2.2. Mycoparasite screening Three replicate plates of each of the Rosellinia isolates and four mycoparasites T. harzianum (ARB4 and T-22), Clonostachys rhizophaga (Tehon and Jacobs) Schroers (ARB11), and Clonostachys cf. byssicola Schroers (ARB37) (Schroers, 2001) were used to measure the effect of pH and phosphate levels on radial growth rates. PDA was adjusted to pH levels of 3–10 (interval 1) using 0.1 M NaOH or 25% v/v lactic acid; the same acid was used within each experiment. The effect of phosphate was measured twice using different phosphate sources. PDA plates were adjusted to phosphate levels of 0.00, 0.01, 0.05, and 0.10 M using K2 HPO4 (J.T. Baker Chemical, Phillipsburg, NJ, USA) and NaH2 PO4 (Merck) and pH values of autoclaved media were measured using a pH-meter (analog pH-meter/model 301, Orion Research, Cambridge MA, USA) and reconfirmed using pH-paper after settling. Plates were incubated in constant darkness at 26  2 °C. The mycoparasites from the first isolation were used in a hostrange experiment. Hostranges of the mycoparasites were determined as described by Krauss et al. (1998) using Rosellinia strains RB1 and RB2 as hosts. Based on their broader hostrange, mycoparasites C. rhizophaga (ARB11) and T. harzianum (ARB4 and T22) were selected for further evaluation in bioassays. For the bioassays, soil was collected in the same field where the Rosellinia isolates from cocoa and their mycoparasites had been obtained. The soil was from a disease-free area but was not sterilized. A sub-sample of the soil was chemically analyzed by CATIEÕs soil science laboratory. The soil was distributed in 2 L seedling bags with a content of 2 kg soil for each bag. Rosellinia was applied at a dose of 250 g colonized wheat seeds per 2 kg soil. The antagonists were applied either singly or as mixture at the same time as the pathogen at a dose of 0.5 L of 1  106 spores ml
1 per 2 kg of soil. When applied as a mixture, the total concentration of the antagonists was kept constant with equal proportions. Five cocoa seedlings were used as replicates for each treatment. Plants were checked weekly and scored according to a nonparametric scale ranging from 0 (healthy plant) to 5

213

(dead plant) (increment 1) to assess disease severity. Dead plants were carefully uprooted and the following parameters were measured: Days of survival, plant height, dry mass of roots, root length, dry mass of leaves, leaf area, and number of leaves. Plant material was dried at 65 °C for two days. Days of survival were calculated from the onset of the experiment until the day the plant was removed. At the end of the assay, all remaining plants were removed and the same parameters were measured. Mortality data were obtained at the end of the experiment by counting for each treatment the number of dead and alive plants. These data were binary, 0 meaning a live plant, 1 a dead plant. A total of five bioassays were carried out. The first two bioassays assessed biocontrol potential of mixed or individual mycoparasites from the first and second collection against Rosellinia. The most effective treatments were selected for the next three bioassays which evaluated the effect of O.M., pH, and phosphate on the success of mycoparasite mixtures to control Rosellinia, respectively. The pH level of the soil was measured using the methodology of Diaz and Hunter (1978). The Cabiria soil had a pH level of 4.2 (Table 2). Three pH levels were used pH 4 (no amendment), pH 5 and 6 were adjusted with calcium carbonate. The O.M. content was measured using the humid digestion method of Walkley and Black (Bornemiza et al., 1979). The soil had 6.1% O.M. (Table 2). Three levels of O.M. were used 6, 9, and 12%. Levels were adjusted to 9 and 12% O.M. using bokashi, a compost material prepared from soil and chicken manure, based on the recipe of Sasaki (1994). For the plant growth parameter root length we used the tap root, measured manually, as a representative but easier to assess parameter. The soil from Cabiria had a high available phosphorus (P) content (Table 2). Therefore, for the phosphate assay, a different soil of low available P content was collected on CATIEÕs experimental farm in San Juan Sur. A sub-sample was also analyzed chemically. The soil had an available phosphate content of 2:3 mg kg
1 (Table 2). Three different phosphate levels were used, 0, 100, and 1000 mg kg
1 . Phosphate was added weekly in the form of NaH2 PO4 (Merck) dissolved in tap water, starting one week before the bioassay at a dose of 200 ml kg
1 soil. During the bioassay, it was added at a rate of 100 ml kg
1 of soil once a week. At the end of the bioassay, available phosphate and soil

Table 2 Soil chemical characteristics of soil collected in the cocoa germplasm collection, Cabiria, and in San Juan Sur, CATIE, Turrialba, Costa Rica Sample

Depth (cm)

pH water

Ca (mg L
1 )

Mg (mg L
1 )

K (mg L
1 )

Pavailable ðmg L
1 Þ

Ptotal ðmg L
1 Þ

Cu (mg L
1 )

Zn (mg L
1 )

Mn (mg L
1 )

O.M. (%)

Cabiria San Juan Sur

0–30 0–80

4.2 4.8

256 28

108 4.8

234 19.5

38.9 2.3

1714 871

23.7 7.7

4.9 0.5

27.9 3.2

6.1 9.4

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ıa et al. / Biological Control 27 (2003) 210–227

pH were determined for all treatments. Instead of T. harzianum ARB4, we included C. cf. byssicola ARB37, an antagonist that showed promising results in our second bioassay. As this mycoparasite was not used before, it was applied individually and in mycoparasite mixtures. Total root length was measured using the computer program WinRHIZO (WinRHIZO v. 3.9a, Regent Instruments, Quebec, Canada, connected to a Hewlett Packard ScanJet 6100C/T); all other parameters were recorded as described before. 2.3. Statistical analysis Radial growth rates were analyzed using one-way analysis of variance (ANOVA). The addition of phosphate altered the pH of the medium; therefore, pH was computed as covariance. Data from the hostrange study were analyzed using the studentÕs t test; differences in susceptibility of the hosts to mycoparasitsm were analyzed using the paired t test. Data from the bioassays to screen effectiveness of mycoparasites were analyzed by one-way ANOVA. Data from the bioassays with the various pHs and levels of O.M., and phosphate were analyzed using two-way ANOVA. Significant treatment effects were determined by TukeyÕs test ðP ¼ 0:05Þ on SAS (SAS, 1985). Mortality data were analyzed using the Cochran-test for binary data and severity was analyzed using the Kruskal–Wallis test, both according to Zar (1996). Percentage mortality from the pH, O.M., andpphosphate assays was arcsine transformed (y 0 ¼ arcsine y, where y is the proportion ranging from 0 to 1), followed by two-way ANOVA. Severity data were compared as area under the disease progress curve (SAS, 1985). As phosphate additions altered the pH of the soil in the phosphate bioassay as well as the amount of available phosphate, pH and available phosphate were computed as covariances. The addition of O.M. had no immediate effect on soil pH. Soil pH was not measured at the end of the O.M. experiment.

3. Results 3.1. Mycoparasite isolation A total of 45 different mycoparasites were collected, of which 43 were found invading precolonized plates with Rosellinia, 11 from the first isolation, and 32 from the second. Apart from these isolates, T. harzianum T-22 and T. virens GL-21 were included in the list of possible antagonists. Three genera made up almost 75% of all mycoparasites: Trichoderma spp. (20, 43.5%), Clonostachys spp. (9, 19.6%), and Fusarium spp. (4, 8.7%). A list of all mycoparasites used in this study and their origin is shown in Table 1.

3.2. Growth optima of pathogens and antagonists Rosellinia and mycoparasite growth rates were determined for all strains on PDA with different pH (Table 3) and phosphate levels (Tables 4a and 4b). All four Rosellinia isolates exhibited growth at all pH levels, except for isolate RB1 which was incapable of growing at pH 3. Optimum pH levels for the four isolates were: pH 4 for isolate RB1, pH 6–8 for RB2, pH 4–6 for RB3, and pH 4–5 for RB4. All mycoparasites grew at all pH levels. T. harzianum ARB4 grew optimally at pH 4 and T-22 at pH 6; C. rhizophaga ARB11 showed no pronounced optimum but had the slowest growth rate at pH 4; C. cf. byssicola ARB37 grew optimally at pH 6–7 (Table 3). Dipotassium phosphate ðK2 HPO4 Þ increased the pH of the medium by up to 1.5 units with increasing phosphate levels (Table 4a). In contrast, monosodium phosphate ðNaH2 PO4 Þ decreased the pH value by 0.5 units with the same increase in phosphate concentration (Table 4b). Given the effect of pH on growth of the fungi tested (Table 3), the ANOVA for phosphate concentration was adjusted for pH as a covariance. The test fungi grew on the PDA plates at all phosphate levels. With K2 HPO4 as phosphate source (Table 4a), isolate RB1 grew significantly ðP < 0:05Þ faster on plates with phosphate than on plates with no added phosphate. Isolate RB2 showed no significant differences in growth rates. Isolates RB3 and RB4 showed declining growth rates with increasing phosphate levels and grew fastest in the absence of phosphate. C. rhizophaga (ARB11) had the highest growth rate at K-phosphate level 0.01 M, whereas growth rates of C. cf. byssicola (ARB37) were not significantly different. T. harzianum (T-22) grew significantly faster with decreasing potassium phosphate levels. With NaH2 PO4 as a phosphate source (Table 4b), results were different for all fungi except C. cf. byssicola (ARB37), which was insensitive to phosphate levels in either form. Growth rates of Rosellinia RB1 and RB4 did not differ between sodium phosphate levels. Isolate RB2 grew fastest in the absence of Na-phosphate and RB3 had an optimum growth rate at phosphate levels 0.01–0.05 M. C. rhizophaga (ARB11) grew most on plates with no added phosphate, and T. harzianum (T22) showed an optimum at 0.05 M Na-phosphate. 3.3. Screening of antagonists The first 11 isolated antagonists and the two commercial isolates were subjected to a hostrange experiment using Rosellinia strains RB1 and RB2 as hosts. The results are shown in Table 5. All antagonists except the unidentified ARB10 and the commercial isolate T. virens GL-21 exhibited mycoparasitism against both pathogen isolates. Isolate RB1 was significantly ðP < 0:05Þ more susceptible than RB2.

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Table 3 Growth rates ðmm day
1 Þ of Rosellinia spp. and selected antagonists on PDA with different pH levels under constant darkness Isolate

pH level

Rosellinia sp. RB1 RB2 RB3 RB4 Clonostachys rhizophaga ARB11 Clonostachys cf. byssicola ARB37 Trichoderma harzianum ARB4 T-22 a;b;c;d;e

3

4

5

6

7

8

9

10

0.0a 1.1a 7.2c 13.1d

3.0c 4.9b;c 14.6e 15.1e

1.4b 5.6c;d 14.6e 14.3e

1.1b 6.3d;e 14.5e 13.0d

1.4b 6.8e 10.5d 11.9c

1.0b 6.4e 9.5d 11.0c

1.2b 5.5b;c 3.8b 9.1b

1.4b 4.8b 0.8a 7.5a

2.0a;b

1.8a

2.7b

2.6b

2.7b

2.7b

2.7b

2.7b

1.8a

1.9a;b

2.8d

3.4e

3.1e

2.3c

2.2b;c

2.1a;b;c

15.6d;e 15.4d;e

16.4e 16.3e

15.1d;e 15.2d;e

10.8c 10.4c

7.7b 8.8c

5.4a 6.8b

14.5d 14.4d

18.1f 1.4a

Values in a row followed by the same letter do not differ at P ¼ 0:05.

Table 4a Growth rates ðmm day
1 Þ of Rosellinia spp. and selected antagonists on PDA with different dipotassium phosphate ðK2 HPO4 Þ levels (mol L
1 ; mg kg
1 in parentheses) adjusted for changing pH-value Phosphate concentration:

0.00 M ð0 mg kg
1 Þ

0.01 M ð950 mg kg
1 Þ

0.05 M ð4750 mg kg
1 Þ

0.10 M ð9500 mg kg
1 Þ

pH:

5.0

6.0

6.5

6.5

Isolate Rosellinia sp. RB1 RB2 RB3 RB4

1.60a 3.65a 13.61c 11.64c

2.20b 3.83a 11.56b 8.11b

2.25b 3.08a 10.50a;b 5.81a

2.04b 3.26a 9.75a 4.78a

Clonostachys rhizophaga ARB11

2.83c

2.90d

2.75b

2.68a

Clonostachys cf. byssicola ARB37

1.91a

2.06a

2.35a

2.17a

13.19d

11.47c

5.59b

4.00a

Trichoderma harzianum T-22 a;b;c;d

Values in a row followed by the same letter do not differ at P ¼ 0:05 (ANOVA adjusted for covariance pH).

Table 4b Growth rates ðmm day
1 Þ of Rosellinia spp. and selected antagonists on PDA with different monosodium phosphate ðNaH2 PO4 Þ levels (mol L
1 ; mg kg
1 in parentheses) adjusted for changing pH-values Phosphate concentration:

0.00 M ð0 mg kg
1 Þ

0.01 M ð950 mg kg
1 Þ

0.05 M ð4750 mg kg
1 Þ

0.10 M ð9500 mg kg
1 Þ

pH:

5.0

5.0

4.5

4.5

Rosellinia sp. RB1 RB2 RB3 RB4

3.44a 3.79b 12.61a;b 12.72a

3.33a 1.87a 13.83b 10.58a

3.26a 2.80a;b 13.42b 11.61a

3.44a 2.13a;b 11.44a 11.50a

Clonostachys rhizophaga ARB11

3.67c

3.44b

2.97a

2.83a

Clonostachys cf. byssicola ARB37

2.60a

2.60a

2.75a

2.81a

16.08a;b

15.25a

16.34b

15.36a

Trichoderma harzianum T-22 a;b;c

Values in a row followed by the same letter do not differ at P ¼ 0:05 (ANOVA adjusted for covariance pH).

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Table 5 Growth rates ðmm day
1 Þ for 14 mycoparasites growing on two different Rosellinia isolates, RB1 and RB2 Mycoparasites

Isolates of Rosellinia RB1

RB2

ARB1 ARB2 ARB3 ARB4 ARB5 ARB6 ARB7 ARB8 ARB9 ARB10 ARB11 T-22 GL-21

8:2  0:6 7:9  1:4 5:2  0:5 3:1  1:2 6:9  0:3 6:2  0:7 5:1  0:4 6:4  0:3 1:3  0:2 0:0  0:0 3:0  0:6 9:9  0:7 0:0  0:0

1:6  0:3 1:9  0:3 1:8  0:7 1:6  0:1 1:0  0:1 2:0  0:2 1:9  0:4 0:9  0:3 0:1  0:0 0:0  0:0 3:0  0:3 1:4  0:1 0:0  0:0

Average

4:9  0:88

1:3  0:25

t Value

Probabilitya

8.83 4.12 3.96 1.24 18.04 5.62 6.13 13.24 5.54 n.a.c 0.12 11.34 n.a.

0.002 0.007 0.008 0.142 n.s.b 0.000 0.002 0.002 0.000 0.003 n.a. 0.456 n.s. 0.000 n.a.

4.49d

<0.001

a

According to student t test, critical value ¼ 3.75, **P < 0:01, ***P < 0:001. n.s., not significant. c n.a., not applicable. d Paired t test, critical value ¼ 1.78. b

Of the 12 mycoparasites tested, only two, T. harzianum (ARB4) and C. rhizophaga (ARB11), showed no significant differences with respect to growth rates on both Rosellinia isolates. Due to their broader hostrange, they were selected for use in the bioassays. For comparison, the better of the two commercial strains, T. harzianum (T-22), was included in the bioassays, as T. virens GL-21 did not parasitise Rosellinia. The results of the soil analyses are shown in Table 2. The soil from Cabiria had a pH of 4.2, contained 6.1% O.M. and 38:9 mg kg
1 available phosphate. This soil was used for all bioassays except for the assay with different phosphate levels. Soil from San Juan Sur was employed for the latter. The soil from San Juan Sur had a pH of 4.8 and 9.4% O.M. It contained 2:3 mg kg
1 available phosphate and had a high capacity of phosphate immobilization as indicated by a relatively high total phosphate content of 871 mg kg
1 (Table 2). The pathogenicity of Rosellinia isolate RB1 to cocoa seedlings at the inoculum rate used was determined in a preliminary experiment. Cocoa seeds as well as 3month-old cocoa seedlings were attacked by RB1 within the time span of one month (data not shown). Table 6 shows the results of the first bioassay. Mortality data, disease severity, and days of survival exhibited a consistent tendency: antagonist mixtures at the same total inoculum level controlled Rosellinia better than single strains. The exception was the commercial T. harzianum T-22 which controlled Rosellinia as a single strain as efficiently as the best mixtures. Mycoparasites ARB4, ARB11 and the mixture of ARB4 + T-22 did not significantly differ from the diseased control with respect to all parameters. The mixture with three antagonists

(ARB4 + ARB11 + T-22) performed best and did not differ from the healthy control for any parameter measured. The second best treatment was the mixture of ARB11 + T-22 which was always better than the diseased control and in the range of the healthy control except for leaf area. In the second bioassay, single strains from the second isolation were pre-screened. The results are shown in Table 7. C. cf. byssicola (ARB37) exhibited the greatest biocontrol. It was significantly different from the healthy control only with respect to mortality. It was significantly different from the diseased control for all parameters. The most effective Trichoderma sp. (ARB33) was significantly worse than the healthy control for mortality, leaf area and dry mass of leaves. Treatment ARB33 was significantly worse than ARB37 for leaf area, number of leaves and dry mass of leaves. For all variables measured in the O.M. bioassay, we found significant differences among treatments (Fig. 1, P < 0:001). No significant interactions of treatments with O.M. levels occurred ð0:08 6 P 6 0:93Þ. Both biocontrol treatments were significantly ðP < 0:05Þ better than the diseased control for root dry mass. The mixture of three antagonists did not differ significantly from the healthy control. Additionally, the mixture of two antagonists significantly ðP < 0:05Þ increased plant height as compared with the diseased control. No significant differences were found between both biocontrol treatments. Days of survival ðP ¼ 0:024Þ and root length ðP ¼ 0:022Þ were highest in soils with the lowest percentage of O.M. The other plant growth parameters were not significantly influenced by O.M. However, for mortality and disease severity there was a visible

1

a;b;c;d

Values within a column followed by the same letter are not significantly different (ANOVA followed by Tukey test at P < 0:05, except when stated otherwise). Data on mortality were analyzed using the Cochran-test for binary data. 2 Maximum: 79 days (end of assay). 3 Data on disease severity are analyzed using the Kruskal–Wallis test for non-parametric data.

0.0 0.0a 0.0a 0.0a 0.0a 9.1a;b 25.4b 22.0b 27.8b 29.1a;b 27.6a;b 23.4a;b;c 25.5a;b;c 32.0a 19.5a;b;c 12.1c 16.2b;c 11.9c 2100a;b 3400a 3100a 2900a 2500a 1900a;b;c 800b;c 600c 600c 2700a 2700a 2400a;b 1900a;b;c 2700a 1600b;c 1300c 1200c 1000c 9.4a 10.4a 8.8a;b 8.6a;b 8.8a;b 7.0a;b 5.0b 5.0b 4.6b 116.9a 108.9a;b 91.4b;c 81.9a;b;c 99.9a;b;c 96.5c;d 68.9d 67.3d 66.0d 79.0a 79.0a 79.0a 79.0a 79.0a 67.0a;b 47.0b 35.8b 31.0b 0a 0a 0a 0a 0a 60b 80b 80b 80b Healthy control ARB4 + ARB11 + T-22 ARB11 + T-22 ARB4 + ARB11 T-22 ARB4 + T-22 ARB11 ARB4 Diseased control

23.2a;b 24.9a 23.2a;b 21.1a;b 25.0a 19.5a;b;c 16.9a;b;c 15.7b;c 12.9c

Number of leaves Leaf area ðcm2 Þ Height (cm) Days of survival2 Mortality (%)1 Treatment

Table 6 Effects of different antagonists applied as single strains or in mixtures on control of Rosellinia root rot on cocoa seedlings

Dry mass leaves (mg)

Dry mass roots (mg)

Tap root length (cm)

Disease severity3

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217

tendency that biocontrol efficacy was highest in soils with a low O.M. content and that the mixture of three antagonists performed better than the mixture of two (Fig. 1). In the pH bioassay, treatments differed significantly ðP < 0:001Þ for all parameters. The healthy control differed from all Rosellinia-inoculated treatments (Fig. 2). Except for mortality and dry mass of leaves, the treatment with three mycoparasites was consistently better than the treatment with two antagonists. For days of survival, leaf area, plant height, number of leaves, and dry mass of roots, biological control efficacy of the mixture of three improved with increasing pH. The mixture of two showed a more irregular control behavior. The plant growth factors consistently affected by pH were days of survival ðP ¼ 0:028Þ, disease severity ðP < 0:001Þ, and root dry mass ðP ¼ 0:003Þ. These characteristics augmented with increasing pH. For the variables disease severity, days of survival, plant height, and leaf area we found interactions between pH level and treatment ð0:0006 6 P 6 0:04Þ. Disease severity showed that the mixture of three antagonists was significantly ðP < 0:001Þ better than the diseased control at pH 5 and 6 and better ðP < 0:001Þ than the mixture of two antagonists at pH 6. The mixture of three antagonists had a significantly ðP ¼ 0:03Þ higher number of days of survival as compared with the diseased control and greater plant height ðP < 0:001Þ compared with two antagonists at pH 6. Average available P and soil pH for the three levels (0, 100, 1000 mg kg
1 additions) at the end of the experiment was 4.4, 11, and 54 mg kg
1 available P and 3.1, 2.9, and 3.5 soil pH, respectively. Available phosphate and soil pH were computed as covariates but did not show an association with plant growth parameters. All variables measured in the phosphate bioassay showed significant differences among treatments (Fig. 3). Days of survival and mortality showed some erratic plant damage in single strains ARB11 and ARB37 but no differences between healthy and diseased controls. Days of survival, root length and the three foliar plant growth variables, leaf dry mass, leaf area and number of leaves, were affected by phosphate level (all P < 0:05). In general, these parameters were significantly higher at the highest phosphate level ð1000 mg kg
1 Þ compared with the other two levels, except for days of survival which had an optimum at the intermediate phosphate level ð100 mg kg
1 Þ and was significantly higher ðP ¼ 0:012Þ than the lowest phosphate level ð0 mg kg
1 Þ but did not differ from the highest level ð1000 mg kg
1 Þ. Only for root length did we find a significant interaction between treatments and phosphate ð0:001 6 P 6 0:049Þ. For this parameter, no differences were found between the diseased control and any of the single strain treatments. Treatment ARB37 + T-22 and the mixture of three BCAs had significantly more root length than the diseased control at phosphate level 0 mg kg
1 but not at the

218

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Table 7 Effects of different antagonists applied as single strains on control of Rosellinia root rot in cocoa seedlings Treatment

Mortality1 (%)

Days of survival2

Height (cm)

Leaf area ðcm2 Þ

Number of leaves

Dry mass leaves (mg)

Dry mass roots (mg)

Healthy control ARB12 ARB13 ARB15 ARB16 ARB17 ARB18 ARB19 ARB20 ARB21 ARB22 ARB33 ARB34 ARB35 ARB36 ARB37 GC Diseased control

0a 60d 100f 60d 100f 80e 100f 80e 80e 60d 60d 20b 60d 40c 60d 20b 80e 100f

111.0d 79.0c 48.4a 85.2c 63.6b 81.6c 69.0b;c 79.4c 66.4b 99.0d 86.6c 111.0d 79.0c 102.6d 104.0d 111.0d 69.8b;c 55.0a

16.1c;d 9.1a;b;c 2.2a;b 7.8a;b;c 11.6b;c;d 11.5b;c;d 2.5a;b 7.1a;b;c 1.4a;b 9.5a;b;c 7.0a;b 10.0a;b;c;d 5.7a;b 10.0a;b;c;d 8.9a;b;c 20.4d 3.0a;b 1.2a

548.9b 272.6a 9.9a 119.8a 109.2a 189.2a 23.4a 88.0a 20.6a 137.8a 149.3a 156.0a 98.3a 288.9a 133.6a 612.9b 89.5a 14.2a

7.6c;d 4.8c 1.2a;b 3.0b;c 3.8b;c 3.6b;c 0.6a;b 3.0b;c 0.8a;b 3.8b;c 2.8b;c 5.4c 3.2b;c 5.0c 2.8b;c 9.0d 1.8b 0.5a

980b;c 440a 20a 290a 190a 470a;b 30a 190a 40a 310a 340a 350a 200a 600a;b 260a 1320c 160a 30a

560c;d 440b;c;d 10a 200a;b 190a;b 340a;b;c;d 20a 50a 40a 190a;b 250a;b;c 340a;b;c;d 60a 320a;b;c 210a;b 730d 120a;b 70a

a;b;c;d;e;f Values followed by the same letter are not significantly different (ANOVA followed by Tukey test at P < 0:05, except when stated otherwise). 1 Data on mortality were analyzed using the Cochran-test for binary data. 2 Maximum: 127 days (end of assay).

other two phosphate levels. The healthy controls had a higher root length than the diseased controls at all phosphate levels. The mixture of three was equal to the healthy control, except at phosphate level 100 mg kg
1 where a marked optimum for root length of the healthy control occurred. For the parameters of leaf dry mass, leaf area and plant height, we found that the mixture of all three antagonists performed significantly ðP < 0:05Þ better than the diseased control and did not differ from the healthy control. For leaf dry mass and leaf area, single strain C. rhizophaga (ARB11) also differed significantly ðP ¼ 0:018Þ from the diseased but not from the healthy control. Number of leaves and dry mass of roots showed significant differences between the mixture of three antagonists and the single strain treatment ARB37, the mixture resulting in significantly higher values (P ¼ 0:04 and P ¼ 0:036, respectively) (Fig. 3).

4. Discussion 4.1. Optimization of biocontrol agent Using Rosellinia isolates RB1 and RB2 as hosts, we were able to isolate a total of 43 mycoparasites, most of them belonging to three genera: Trichoderma, Clonostachys, and Fusarium. Trichoderma and Clonostachys spp., especially Clonostachys rosea, formerly known as Gliocladium roseum (Schroers et al., 1999), are well known for their mycoparasitic capabilities and have been used in numerous studies as BCAs of soilborne

pathogens (e.g., Harman, 2000; Papavizas, 1985; Sztejnberg et al., 1987). Fusarium spp. are less known for their mycoparasitic capabilities, although several publications have commented on this (Kwasna et al., 1999; Vajna, 1985). C. rhizophaga is a recently renamed species, which was originally described as Verticillium rhizophaga (Schroers, 2001), an alleged wilt pathogen of elm (Ulmus spp.) (Tehon and Jacobs, 1936). The mycoparasite Pythium oligandrum Drechs. was first isolated from diseased pea roots and implicated with pathogenicity. Only later, it was recognized as a secondary invader, closely following plant infection with Pythium debaryanum Hesse and Pythium ultimum Trow (Drechsler, 1946). Today we know P. oligandrum as one of the most aggressive and fast-acting necrotrophic mycoparasites (Jones and Deacon, 1995) and as commercial BCA (McSpadden Gardener, 2002). It is conceivable that the same phenomenon occurred with C. rhizophaga; the symptoms reported by Tehon and Jacobs (1936) resemble those of Dutch elm disease, caused by Ophiostoma ulmi (Buism.) Nannf. Our study, like many others, showed the potential of these genera to provide BCAs against soilborne plant pathogens but should be followed by an evaluation under field conditions. Results of previous studies on biocontrol of Rosellinia spp. look promising in initial tests (Castro, 1995; Esquivel et al., 1992; Freeman et al., 1986; Ruiz and Leguizam
on, 1996; Sztejnberg et al., 1987). However, subsequent field trials were disappointing (Sztejnberg et al., 1987; Kojima, 1987, cited by Matsumoto, 1998).

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This common phenomenon has been ascribed to the development procedures used to identify potential biocontrol agents and the lack of ecological considerations on target diseases before application (Deacon, 1991). The use of sterilized soils in some of the bioassays may give clearer results, but a good biocontrol agent also gives significant control in non-sterile conditions. Furthermore, application of the large amounts of antagonist inoculum some time before the application of comparatively little pathogen inoculum gives the antagonist a clear advantage as it has time to become established in the soil (Leeman et al., 1996). Therefore, in this study, we assessed biocontrol efficacy under severe disease pressure and under non-sterile conditions. Inoculation of the antagonist and the pathogen was done simultaneously and the amount of pathogen inoculum was considerably higher than in other published studies (from 2 to almost a 100 times). Under these strict conditions, we prescreened a total of 19 antagonists as single strains. Although the majority of mycoparasites differed significantly from the diseased control, only two, C. cf. byssicola ARB37 and the commercial T. harzianum T-22, did not differ from the healthy control (Tables 6 and 7). The mycoparasite mixtures, except the mixture of T. harzianum strains ARB4 + T-22, were generally better than the individual antagonists and did not differ from the healthy control. The antagonist mixture with three mycoparasites was more effective and consistent than the mixtures of two antagonists (Tables 6 and 7 and Figs. 1–3). Mixed bioinocula for the control of soilborne diseases have been pioneered by Sivasithamparam and Parker (1978) and were reviewed by Raupach and Kloepper (1998). Single antagonists are unlikely to be active in all soil environments in which they are applied, whereas mixed inocula will have a wider range of environmental adaptations such as different pH values, soil O.M., phosphorus, and moisture content. In our study, the mixture of three mycoparasites was less affected by changing pH values and, to a lesser extent, O.M. than the mixture of two (Figs. 1 and 2). Future research should investigate whether mixtures composed of antagonists from the first (dry season) and second (rainy season) isolations exhibit increased survival and efficacy at different water potentials of the soil. Mixed inocula may also possess a variety of disease suppression mechanisms and hence, may control a wider range of pathogens than individual strains. The Rosellinia strains used in this study exhibited significant differences in their susceptibility to mycoparasitsm (Table 5). In order to design a more versatile bioinoculum, future bioassays should employ mixtures of the different pathogen strains. However, Harman (2000) suggests that some individual strains, such as T. harzianum T-22, can also exhibit biocontrol of various unrelated fungal pathogens in a wide range of similarly unrelated crops and orna-

219

mentals, independent of the soil type and geographic location. Strain T-22 is undoubtedly an exceptionally versatile BCA; yet mixing this with our native isolates still improved its biocontrol efficacy and consistency. Furthermore, strictly speaking, strain T-22 is a strain mixture itself, as it was constructed by protoplast fusion of two highly effective T. harzianum strains, one a rhizosphere competent mutant produced from an antagonist of Rhizoctonia solani K€ uhn of Colombian origin, and the other an antibacterial strain with high tolerance to iron-limiting conditions from the US (Harman, 2000). We, therefore, conclude that strain mixtures may be the simplest approach to increase the likelihood of obtaining a versatile and consistently effective BCA against soilborne pathogens. However, incompatibility between antagonists has been reported (Raupach and Kloepper, 1998). The mixture of T-22 + ARB4 in our study was intermediate between the individual antagonists; no synergistic effect was observed. Further investigations on compatibility between antagonists seem therefore warranted. 4.2. Organic matter Organic matter amendments are used in the management of a wide range of soilborne diseases. Even with pathogens which thrive on crop residues, such as R. solani or Macrophomina phaseolina (Tassi) Goid., organic amendments usually reduced disease. Furthermore, they often have a synergistic effect on biocontrol (Baby and Manibhushanrao, 1993; Boosalis, 1956; Papavizas and Lumsden, 1980; Raguchander et al., 1998). Rosellinia seems an exception in that root rot is consistently associated with areas rich in O.M. (Aranzazu et al., 1999; Ruiz and Leguizam
on, 1996; Stell, 1929; Waterston, 1941). With increasing O.M. in our study, biocontrol efficiency decreased, while mortality and disease severity increased (Fig. 1). This is in concordance with the results of Ruiz and Leguizam
on (1996) who found higher infection levels in soils with 8.9% O.M. than soils with 6.2 and 3.5% O.M., although the antagonists were also favored by high O.M. content. We therefore conclude that Rosellinia is more capable of exploiting these resources than the BCAs. Although atypical, these observations are not unreasonable. The frequently reported increase in population levels of Trichoderma spp. as determined by dilution plating, following the addition of O.M. to the soil (Osando and Waudo, 1992; Otieno, 1998) is no indication of enhanced physiological activity of the BCA because dilution plating preferentially detects spores, i.e. resting structures. Organic amendments can break dormancy but the ammonia released during decomposition is fungistatic (Papavizas, 1985). This may help to explain the variability found between different types of organic amendments. Otieno (1998) observed no reduction of Armillaria root rot in tea although indigenous Tricho-

220

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Fig. 1. Effects of soil organic matter content on biocontrol of Rosellinia sp. in cocoa seedlings using mixtures of C. rhizophaga (ARB11) and T. harzianum (ARB4 and T-22).

derma populations were enhanced by the addition of coffee pulp. Casale et al. (1995) even found a negative correlation between the nitrogen content of various mulches and the growth of T. harzianum as well as the percentage of healthy avocado and citrus roots. The deleterious effect of ammonia is more pronounced in alkaline soils, and T. hamatum and T. koningii are more sensitive to it than C. rosea and T. harzianum (Papavizas, 1985).

The fact that in our study the competitive ability of C. rhizophaga and T. harzianum was significantly impaired by O.M. at relatively low pH value suggests that O.M. should be minimized in the management of Rosellinia root rot in cocoa and is in agreement with the recommendation to remove CWD (Aranzazu et al., 1999; Nowell, 1916; Waterston, 1941). Bokashi is a high-nitrogen amendment and the effect of high

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221

Fig. 1. (continued)

C/N amendments on the interaction merits further study. 4.3. Soil pH According to the literature, the effect of soil pH on Rosellinia root rot is less conclusive than the effect of O.M. (Cadavid, 1995; L
opez and Fern
andez, 1966; Waterston, 1941). Nevertheless, various authors have promoted the use of lime to control Rosellinia root rot, as acid soils are said to favor the development of the disease (Aranzazu et al., 1999; Herrera and Grillo, 1989; Nowell, 1916). In our study, pH influenced five of the nine growth parameters assessed, whereas O.M. influenced only two. We therefore conclude that pH is at least as important as O.M. Rosellinia grows under a wide range of pH levels (Table 3) but different isolates have significantly different pH optima. Our findings corroborate results by Waterston (1941), and L
opez and Fern
andez (1966) that R. pepo as well as R. bunodes grew under a wide range of pH levels (pH 3–9) on artificial media. In the bioassay with different levels of soil acidity, mortality was 100% for the diseased control as well as the two antagonist treatments (Fig. 2),

implying that Rosellinia is not limited in its capacity to attack cocoa at pH 4–6. A significant interaction with disease severity indicated that biocontrol efficacy depends on the antagonist mixture and the pH. At pH 4, biocontrol was ineffective. However, at pH levels 5 and 6, the mixture of three antagonists was significantly better than the diseased control. Rosellinia strain RB1 has a pH optimum of 4 in vitro (Table 3) which coincides with the pH of the soil of origin (Table 2). It would be worthwhile to investigate whether this trend is reversed if a pathogen isolate with a high pH optimum is employed in the bioassay instead. In this respect the potato strain RB2 may be a useful tool. Like Rosellinia spp., Trichoderma spp. are favored by acid soils (Casale et al., 1995; Mondal et al., 1996; Papavizas, 1985; Singh et al., 1998) and liming was found to be detrimental to T. harzianum and its biocontrol efficacy against R. solani (Mu~ noz et al., 2001). However, Trichoderma spp. and C. rosea have been isolated from soils ranging from pH 4 to 8.5 (Foley and Deacon, 1985; Wen et al., 1993) and even with T. harzianum, the pH optimum varies greatly between strains (Singh et al., 1998). Our native T. harzianum ARB4 had a pH optimum of 4, coinciding with its soil

222

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Fig. 2. Effects of soil pH level on biocontrol of Rosellinia sp. in cocoa seedlings using mixtures of C. rhizophaga (ARB11) and T. harzianum (ARB4 and T-22).

of origin, whereas the commercial strain T-22 grew best at pH 6 (Table 3), the upper limit of pH levels tested in the greenhouse. Our C. rhizophaga showed no pH preference. This may explain why, for most parameters, biocontrol efficacy of the mixture of three mycoparasites increased with increasing pH, whereas the mixture of two antagonists showed a much more irregular control behavior. The more complex mixture

had a stabilizing effect on biocontrol success by providing a wider range of environmental circumstances under which it could work (Raupach and Kloepper, 1998). Cocoa is capable of growing well in soils ranging from pH 4.5 to 8, and with an optimum pH level of about 6.5 (Mossu, 1990). Based on our results, we can conclude that liming is not only compatible with biocontrol but probably synergistic, as biocontrol efficacy

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223

Fig. 2. (continued)

was enhanced with increasing pH levels. It may even be possible to further improve the biocontrol mixture for an integrated management approach with liming by incorporating an antagonist with a high pH optimum into the inoculum. 4.4. Phosphate The effect of phosphate on growth rates of the Rosellinia isolates and the mycoparasites (Tables 4a and 4b) depended on the phosphate source. Both affected five out of seven isolates. C. cf. byssicola (ARB37) was not affected, whereas RB2 reacted to monosodium but not to dipotassium phosphate; the reverse was true for RB1. Dipotassium phosphate had a larger effect on the pH level of the medium than monosodium phosphate which may explain the reaction of Rosellinia isolates RB2 and RB4 but not of RB1 and RB3, C. rhizophaga ARB11 and T. harzianum T-22 (Tables 3, 4a and 4b). L
opez and Fern
andez (1966) stated that phosphate concentrations of 0.05–0.1 M considerably reduced growth of R. pepo and R. bunodes but did not present any data. Monopotassium phosphate is a fungicide against powdery mil-

dews (EPA, undated-a) and dipotassium phosphate is currently under investigation as a fungicide (EPA, undated-b). However, together with the results from the monosodium phosphate growth experiment it seems likely that potassium levels (0, 0.02, 0.1, and 0.2 M) rather than phosphate inhibit growth. The individual effects of phosphate, sodium and potassium ions on Rosellinia and its mycoparasites merit further investigation. At the end of the phosphate bioassay, available P had increased in the treatments with the weekly additions of 100 and 1000 mg kg
1 monosodium phosphate. Soil pH was markedly decreased for all treatments at all phosphate levels. Altomare et al. (1999) found that T. harzianum T-22 is capable of solubilizing rock phosphate but did not produce organic acids. However, in our study, no effect on phosphate was detected in treatments that included T-22. The effect of phosphate on Rosellinia root rot was the least conclusive of the three soil characteristics investigated. Foliar plant growth parameters increased with increasing phosphate levels, whereas root length decreased and root dry mass exhibited an optimum at the intermediate phosphate level. An explanation could be

224

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Fig. 3. Effects of soil phosphate level on biocontrol of Rosellinia sp. in cocoa seedlings using single strains and mixture of C. rhizophaga (ARB11), C. cf. byssicola (ARB37) and T. harzianum (T-22).

that the scarceness of phosphate allows for the development of many fine roots to search for available phosphate, hence higher root length but lower dry mass. Possibly, the only role of phosphate in Rosellinia root rot is that phosphate aids plant growth and vigor, thereby rendering infection by an opportunistic pathogen more difficult.

Waterston (1941) and Cadavid (1995) stated that Rosellinia prevails in soils with available P levels below 40 mg kg
1 . Tropical soils, though, rarely reach available P levels of 40 mg kg
1 (Dabin, 1980) even in heavily fertilized areas. Furthermore, tropical soils low in P are generally acidic which favors Rosellinia. The amount of phosphate added in this experiment ranged between 0 and

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225

Fig. 3. (continued)

1000 mg kg
1 . This was done in order to detect any possible effects of P, even if the addition of these phosphate levels is not realistic in the field. We therefore conclude that, within a realistic range for cocoa growth, phosphate is of little importance to the equilibrium between Rosellinia and its BCAs compared to pH and O.M.

interactions in the complex soil ecosystem, compatibility of mycoparasites, and the possible interactions between cultural controls consisting of liming, removal of CWD and biological control under field conditions.

Acknowledgments 4.5. Future prospects Opportunistic soilborne pathogens such as Rosellinia spp. are notoriously difficult to control once they manifest themselves because when the first symptoms appear, the pathogen is well established in soil and roots. Biocontrol tends to be preventative rather than curative. Together with delivery problems, this may explain the lack of success under field conditions to date (Sztejnberg et al., 1987). Realistically, the main scope for biocontrol and liming is in the replanting of affected areas after dead and decomposing material has been cleared. Our results suggest that biocontrol mixtures can be effective in controlling Rosellinia root rot in cocoa. Further investigations are needed on pathogen-antagonist

The authors gratefully acknowledge funding by USDA-ARS, DGIS (MtH), and NORAD (RMG) managed by CABI Bioscience and CATIE. The authors thank their colleagues at CATIE and CABI for their support and useful discussion, especially Ghiselle Alvarado, Claudio Arroyo, John Beer, Eduardo Brenes, Harry Evans, Julie Flood, Andr
e George, Eduardo Hidalgo, Adolfo Mart
ınez, Maribel Mora, Johnny P
erez, Armando Portuguez, Carla Salmer
on, Miguel Sanabria, Andrea Schl€ onvoigt, Michelle Schroeder, and Tim Stirrup. The authors would especially like to thank Hans-Josef Schroers (CBS) for identifying our Clonostachys spp. and useful information on C. rhizophaga. The mentioning of trade names does not constitute a recommendation for the product.

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References Achicanoy, H., 1989. Sensibilidad de Rosellinia pepo Pat. causante de la llaga estrellada del cacao (Theobroma cacao L.) a funguicidas in vitro. In: Abstracts of the Congress of the Asociaci
on Latinoamericana de Fitopat
ologos, the American Phytopathological Society—Caribbean Division and the Asociaci
on Colombiana de Fitopat
ologos y Ciencias Afines. CIAT, Cali, Colombia, 10–14 July, 1989, p. 38. Altomare, C., Norvell, W.A., Bj€ orkman, T., Harman, G.E., 1999. Solubilization of phosphates and micronutrients by the plantgrowth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Appl. Environ. Microbiol. 65, 2926–2933. Aranzazu, H.F., 1996. Comportamiento de la llaga estrellada Rosellinia pepo Pat. sobre raices de cacao. Fitopatolog
ıa Colombiana 20, 7–10. Aranzazu, H.F., 1997. Control de la llaga estrellada en cacao causada por Rosellinia pepo Pat. Fitopatalogia Colombiana 21, 5–9. Aranzazu, H.F., C
ardenas, L.J., Mujica, J.J., G
omez, Q.R., 1999. Manejo de las llagas radicales (Rosellinia sp.). In: Boletin de Sanidad Vegetal 23, Instituto Colombiano Agropecuario (ICA) and Corpoica, Santaf
e de Bogot
a, Colombia, p. 35. Baby, U.I., Manibhushanrao, K., 1993. Control of rice sheath blight through the integration of fungal antagonists and organic amendments. Trop. Agric. 70, 240–244. Bautista, P.F., Magdiel, S.M., 2000. Evaluaci
on de da~ nos econ
omicos causados por Rosellinia sp. en un
area afectada por el pat
ogeno. Available from (accessed April 30, 2002). Bautista, P.F., Rivera, M.A.A., 1997. Evaluaci
on de agroqu
ımicos para combatir ataques de (Rosellinia sp.) en el cultivo del caf
e. In: Echeverri, J., Mora, O., Zamora, L. (Eds.), Memorias. 18. Simposio Latino-Americano de Caficultura. IICA, Promecaf
e, Instituto de Caf
e de Costa Rica, San Jos
e, Costa Rica, 16–19 September 1997, pp. 339–343. Boosalis, M.G., 1956. Effect of soil temperature and green-manure amendment of unsterilized soil on parasitism of Rhizoctonia solani by Penicillium vermiculatum and Trichoderma sp. Phytopathology 46, 473–478. Booth, C., Holliday, P., 1972. Rosellinia pepo. In: Descriptions of Pathogenic Fungi and Bacteria, vol. 354. Commonwealth Mycological Institute, Kew, Surrey, England. Bornemiza, E., Constenla, M., Alvarado, A., Ortega, E.J., V
asquez, A.J., 1979. Organic carbon determination by the Walkley-Black and dry combustion methods in surface soils and depth profiles from Costa Rica. Soil Sci. Soc. Am. J. 43, 78–83. Cadavid, S., 1995. Rosellinia in cocoa. Cocoa GrowersÕ Bull. 49, 52–59. Casale, W.L., Minassian, V., Menge, J.A., Lovatt, C.J., Pond, E., Johnson, E., Guillernet, F., 1995. Urban and agricultural wastes for use as mulch on avocado and citrus and for delivery of microbial biocontrol agents. J. Horticult. Sci. 70, 315–332. Castro, B.L., 1995. Antagonismo de algunos aislamientos de Trichoderma koningii, originados en suelo Colombiano contra Rosellinia bunodes, Sclerotinia sclerotiorum y Pythium ultimum. Fitopatolog
ıa Colombiana 19, 7–18. Cubillos, G., 1988. Eficiencia de campo del fungicida sist
emico tridemorph en el control de la Rosellinia del cacao. El Cacaotero Colombiano 11, 27–33. Dabin, B., 1980. Phosphorus deficiency in tropical soils as a constraint on agricultural output. In: Asda, A. (Ed.), Priorities for Alleviating Soil-Related Constraints to Food Production in the Tropics. International Rice Research Institute, Manila, Philippines, pp. 217–232, p. 468. Deacon, J.W., 1991. Significance of ecology in the development of biocontrol agents against soil-borne plant pathogens. Biocontrol Sci. Technol. 1, 5–20.

Diaz, R.R., Hunter, A., 1978. Metodolog
ıa de Muestreo de Suelos, An
alisis Qu
ımico de Suelos y Tejido Vegetal e Investigaci
on en Invernaderos. CATIE, Turrialba, Costa Rica, 65. Drechsler, C., 1946. Several species of Pythium peculiar in their sexual development. Phytopathology 36, 781–864. EPA, undated a, Available from (accessed April 30, 2002). EPA, undated b, Available from (accessed April 30, 2002). Esquivel, R.V.H., Leguizam
on, C.J.E., Arbelaez, T., 1992. B
usqueda y evaluaci
on de antagonistas a Rosellinia bunodes agente causante de la llaga negra del cafeto. Cenicaf
e (Colombia) 43, 33–42. Feitosa, M.J., Pimentel, C.P.V., 1991. Rosellinia buno des (Berk. et Br.) Sacc., fungo patog^enico a cacaueiros (Theobroma cacao L.) no estado de S~ao Paulo. Cient
ıfica, S~ao Paulo 19, 31–35. Foley, M.F., Deacon, J.W., 1985. Isolation of Pythium oligandrum and other necrotrophic mycoparasites from soil. Trans. Br. Mycol. Soc. 85, 631–639. Freeman, S., Sztejnberg, A., Chet, I., 1986. Evaluation of Trichoderma as a biocontrol agent for Rosellinia necatrix. Plant and Soil 94, 163–170. Harman, G.E., 2000. Myths and dogmas of biocontrol. Changes in perceptions derived from research on Trichoderma harzianum T-22. Plant Dis. 84, 377–393. Herrera, I.L., Grillo, R.H., 1989. La pudrici
on negra de las ra
ıces del cafeto en la regi
on del Escambray. Revista Centro Agr
ıcola (Cuba) 16, 53–59. Jones, E.E., Deacon, J.W., 1995. Comparative physiology and behaviour of the mycoparasites Pythium acanthophoron, P. oligandrum and P. mycoparasiticum. Biocontrol Sci. Technol. 5, 27–39. Krauss, U., Bidwell, R., Ince, J., 1998. Isolation and preliminary evaluation of mycoparasites as biocontrol agents against crown rot of banana. Biol. Control 13, 111–119. Kwasna, H., Dawson, W.A.J.M., Bateman, G.L., 1999. Mycoparasitsm of Coemansia species. Mycol. Res. 103, 925–928. Leeman, M., den Ouden, F.M., van Pelt, J.A., Cornelissen, C., Matamala-Garros, A., Bakker, P.A.H.M., Schippers, B., 1996. Suppression of Fusarium wilt of radish by co-inoculation of fluorescent Pseudomonas spp. and root-colonizing fungi. Eur. J. Plant Pathol. 102, 21–31. L
opez, D.S., Fern
andez, B.O., 1966. Llagas radicales negra (Rosellinia bunodes) y estrellada (Rosellinia pepo) del cafeto. II. Effecto de la humedad y pH del suelo en el desarrollo micelial e infecci
on. Cenicaf
e (Colombia) 17, 61–69. Lumsden, R.D., Walter, J.F., Baker, C.P., 1996. Development of Gliocladium virens for damping-off disease control. Can. J. Plant Pathol. 18, 463–468. Matsumoto, N., 1998. Biological control of root diseases with dsRNA based on population structure of pathogens. Jpn. Agric. Res. Quarterly 32, 31–35. McSpadden Gardener, B., 2002. Commercial biocontrol products available for use against plant pathogens. Available from (accessed May 29, 2002). Merchan, V.M., 1989. Manejo de enfermedades en cacao. Ascolfi Inform. 15, 10–14. Mondal, G., Srivastava, K.D., Aggarwal, R., Singh, D.V., 1996. Population dynamics of Trichoderma viride and T. koningii under different ecological conditions. Indian J. Microbiol. 36, 165–166. Mossu, G., 1990. In: Le Cacaoyer. Maisonneuve et Larose, Paris, p. 160. Mu~ noz, R.C., Virgen, C.G., Herrera, E.A., Olalde, P.V., 2001. Introducci
on de agentes de control biol
ogico de Rhizoctonia solani en suelos solarizados o encalados en condiciones de invernadero. Manejo Integrado de Plagas 59, 10–14.

R.A. Mendoza Garc
ıa et al. / Biological Control 27 (2003) 210–227 Nowell, W., 1916. Rosellinia root diseases in the Lesser Antilles. West Indian Bull. 16, 31–77. Osando, J.M., Waudo, S.W., 1992. Effect of coffee pulp on Trichoderma spp. in Kenyan tea soils. Trop. Pest Manage. 38, 376–381. Otieno, W., 1998. Armillaria-Trichoderma interaction and management of Armillaria root rot of tea (Camellia sinensis (L.) O. Kuntze). Tea 19, 11–16. Papavizas, G.C., 1973. Status of applied biological control of soilborne plant pathogens. Soil Biol. Biochem. 5, 709–720. Papavizas, G.C., 1985. Trichoderma and Gliocladium: biology, ecology, and potential for biocontrol. Ann. Rev. Phytopathol. 23, 23–54. Papavizas, G.C., Lumsden, R.D., 1980. Biological control of soilborne fungal propagules. Annu. Rev. Phytopathol. 18, 389–413. Raguchander, T., Rajappan, K., Samiyappan, R., 1998. Influence of biocontrol agents and organic amendments on soyabean root rot. Int. J. Trop. Agric. 16, 247–252. Raupach, G.S., Kloepper, J.W., 1998. Mixtures of plant growthpromoting rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 88, 1158–1164. Ruiz, S.L., Leguizam
on, C.J., 1996. Efecto del contenido de materia org
anica del suelo sobre el control de Rosellinia bunodes con Trichoderma spp. Cenicaf
e 47, 179–186. Saccas, A.M., 1956. Les Rosellinia des caf
eiers en Oubangui-Chari. LÕAgronomie Tropicale 11, 551–595; LÕAgronomie Tropicale 11, 687–706. SAS Institute 1985. SAS Users guide: Statistics. SAS Institute, Cary, NC, USA. Sasaki, S., 1994. In: Manual del Curso B
asico de Agricultura Org
anica. Universidad de Costa Rica, San Jos
e, Costa Rica, pp. 4– 21. Schroers, H-J., 2001. A monograph of Bionectria (Ascomycota, Hypocreales, Bionectriaceae) and its Clonostachys anamorphs. Stud. Mycol. 46, 1–214. Schroers, H.-J., Samuels, G.J., Seifert, K.A., Gams, W., 1999. Classification of the mycoparasitic Gliocladium roseum in Clonostachys as C. rosea, its relationship to Bionectria ochraleuca, and notes on other Gliocladium-like fungi. Mycology 91, 365–385. Shirata, A., Tsukamoto, T., Yasui, H., Hata, T., Hayasaka, S., Kojima, A., Kato, H., 2000. Isolation of bacteria producing bluish-

227

purple pigment and use for dyeing. Jpn. Agric. Res. Quarterly 34, 131–140. Singh, R.S., Jagpal Singh, Singh, H.V., 1998. Effect of irrigation and pH on effect of Trichoderma in biocontrol of black scurf of potato. In: Dhaliwal, G.S., Arora, R., Randhawa, N.S., Dhawan, A.K. (Eds.), Ecological Agriculture and Sustainable Development, vol. 2, Proceedings of the International Conference on Ecological Agriculture: Towards sustainable Development. Chandigarh, India, 15–17 November, 1997, Centre for Research and Industrial Development, Chandigarh, India, pp. 375–381. Sivanesan, A., Holliday, P., 1972. Rosellinia bunodes. In: Descriptions of Pathogenic Fungi and Bacteria, vol. 351. Commonwealth Mycological Institute, Kew, Surrey, England. Sivasithamparam, K., Parker, C.A., 1978. Effect of certain isolates of bacteria and actinomycetes on Geaumannomyces graminis var. tritici and take-all off wheat. Aust. J. Botany 26, 773–782. Stell, F., 1929. Annual Report for the Department of Agriculture Trinidad and Tobago for the Year 1927. Port of Spain, Trinidad and Tobago, p. 33. Stockdale, F.A., 1908. Fungus diseases of cacao and sanitation of cacao orchards. West Indian Bull. 9, 166–170. Sztejnberg, A., Freeman, S., Chet, I., Katan, J., 1987. Control of Rosellinia necatrix in soil and in apple orchards by solarization and Trichoderma harzianum. Plant Dis. 71, 365–369. Tehon, L.-R., Jacobs, H.L., 1936. A verticillium root rot of American elm. Bull. Davey Tree Expert Company 6, 3–32. Vajna, J., 1985. Phytopathogenic Fusarium oxysporum Schlecht as a necrotrophic mycoparasite. Phytopathol. Zeitschr. 114, 338–347. Waterston, J.M., 1941. Observations on the parasitism of Rosellinia pepo Pat. Trop. Agric. 18, 174–184. Wen, C.J., Tao, J.F., Cheng, W.R., 1993. Studies on the taxonomy of the genus Trichoderma in southwest China. Acta Mycol. Sini. 12, 118–130. Yoshida, S., Hiradate, S., Tsukamoto, T., Hatakeda, K., Shirata, A., 2001. Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves. Phytopathology 91, 181–187. Zar, J.H., 1996. Biostatistical Analysis, third ed. Prentice-Hall, Englewood Cliff, NJ.