**3.3.1 Effect of** *Trichoderma* **on the mycelial growth of** *P. palmivora*

The direct confrontation tests realized *in vitro*, between the isolates of *Trichoderma* sp. and of those of *Phytophthora palmivora* revealed an inhibitory effect of *Trichoderma* on *P. palmivora* in mixed culture. After 3 days of confrontation, the inhibition of the growth speed became very high and the growth of *P. palmivora* practically stops (Fig.2).

Fig. 2. Influence of *Trichoderma* on the mycelial growth of *Phytophthora palmivora.* Legend. PP : *Phytophthora palmivora*, Tricho: *Trichoderma*, P/Tricho : *Phytophthora/Trichoderma*

The capacity of *Trichoderma* to stop the mycelial growth of *P. palmivora* reveals a deep fungistatic effect. From the fourth day, we note a progressive disappearance of the mycelium of *P. palmivora*. This degradation of the mycelium of *P. palmivora* which is more accentuated at the fifth day, with all the isolates, reveals a mycoparasitic effect of

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in

0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50 4,00 4,50 5,00

Scores of leaf susceptibility

Control

IFC5, P7 and SCA6.

Treatments

*P. palmivora* 

T39

T49

T51

T26

T47

T54

T3

T6

T13

T46

T38

T12

T37

T40

T11

T52

T48

T50

T20

T30

T16

T5

T35

T44

T32

Fig. 4. Effect of *Trichoderma* strains on the leaf discs susceptibility to *P. palmivora* for clones

**3.3.3 Field efficacy of** *Trichoderma* **in the control of the black pod disease due to** 

Rotten pods (%)

Year1 Untreated control 18.33 - 15.6

Year2 Untreated control 7.46 - 12.04

species of *Trichoderma* during 2 years of field trials after the application

The effects of 4 species of *Trichoderma*, applied to the cacao trees were compared. The results obtained are presented in Table 3. The final percentages of rotten pods for year 1, were 11.75, 8.44, 7.34, and 3.6 respectively with the isolates T4 of *T. spirale*, the isolate T40 of *T. harzianum*, the isolate T7 of *T. virens* and the isolate T54 of *T*. *asperellum*. These percentages were lower than 18.33 obtained with the untreated control, indicating a reduction of the yield losses from 36 to 80 %. For year 2, the final percentages of diseased pods were lower than that recorded in year 1. For both years of study, the calculated efficacy index was

> *T. spirale* T4 11.75 36 8.96 42 *T. harzianum* T40 8.44 54 8.82 43 *T. virens* T7 7.34 60 5.29 66 *T. asperellum* T54 3.6 80 3.29 79

> *T. harzianum* T40 4.16 44 6.73 44 *T. virens* T7 4.23 43 6.08 49 *T. spirale* T4 2.46 67 4.81 60 *T. asperellum* T54 2.21 70 4.68 61

Table 3. Reduction of the final losses due to the black pod disease and efficacy (%) of 4

T42

T45

T24

T15

T18

*Trichoderma* strains

T22

T34

T14

T36

Reduction of loss in relation to untreated control (%)

T27

T2

T25

T41

T1

T17

T7

T4

T9

T43

T28

Slope of the epidermic curve

T29

T23

T33

T8

T10

T19

T58

T31

T21

T57

Efficacy index (%)

T56

T53

T55

Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 311

IFC5 P7 SCA6

*Trichoderma*. After 7 days of confrontation, in mixed culture, the survival of the spores of *P. palmivora* was assessed. The influence of *Trichoderma* sp. varied according to isolates. The percentage of survival of *Phytophthora* varied from 90 to 50 % respectively with *Trichoderma* isolates 3 and 6. This rate falls to 30 to 10 % with *Trichoderma* 2 and 4. Finally, the presence of *Trichoderma* 1 and 5 in mixed culture has a very clear fungicidal effect with a percentage of survival of *P. palmivora* equal to 0.

#### **3.3.2 Influence of the bacteria and** *Trichoderma* **on the leaf susceptibility to** *P. palmivora*

The effect of 37 strains of bacteria on *P. palmivora* was evaluated using the leaf disk test. The results showed that, for the resistant clone (SCA 6) of cacao tree, the rating scores of the leaf susceptibility varied from 3.3 to 0.59, respectively with strains B104 and B105 (Fig. 3).

Fig. 3. Effect of the bacterial strains on the leaf discs susceptibility to *P. palmivora* for clones IFC5, P7 and SCA6.

With the moderately resistant clone (P7), the rating scores varied in the same proportions. In both cases, the analysis of variance revealed a significant (P<0.05) effect of bacteria and the Student Newman & Keuls test revealed 5 homogeneous groups of bacteria (a, ab, b, bc, and c). The best results were obtained with bacteria BI05 and B116 (group c). With the susceptible clone IFC 5, we note that the reduction of the rating scores of leaf susceptibility is relatively low. The scores varied from 3.8 to 2.8. The statistical analyses did not reveal any significant (P>0.05) differences between the treatments and the untreated control.

Similarly, the effect of 57 *Trichoderma* isolates on *P. palmivora* was evaluated using leaf disk of three cocoa clones. For the susceptible clone (IFC5), the scores of leaf susceptibility varied from 2.4 to 0.02 respectively with the isolates T39 of *T. spirale* and T55 of *T. virens*. With the moderately resistant clone (P7), we note a reduction of the scores, which varied from 2.04 to 0.03 respectively with the isolates T39 of *T. spirale* and T28 of *T. virens*. With the resistant clone (SCA6), the scores were less than 1.5 for all the isolates of *Trichoderma* (Fig. 4).

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 311

310 Biodiversity Loss in a Changing Planet

*Trichoderma*. After 7 days of confrontation, in mixed culture, the survival of the spores of *P. palmivora* was assessed. The influence of *Trichoderma* sp. varied according to isolates. The percentage of survival of *Phytophthora* varied from 90 to 50 % respectively with *Trichoderma* isolates 3 and 6. This rate falls to 30 to 10 % with *Trichoderma* 2 and 4. Finally, the presence of *Trichoderma* 1 and 5 in mixed culture has a very clear fungicidal effect with a percentage of

The effect of 37 strains of bacteria on *P. palmivora* was evaluated using the leaf disk test. The results showed that, for the resistant clone (SCA 6) of cacao tree, the rating scores of the leaf

IFC5 P7 SCA6

**3.3.2 Influence of the bacteria and** *Trichoderma* **on the leaf susceptibility to** *P.* 

susceptibility varied from 3.3 to 0.59, respectively with strains B104 and B105 (Fig. 3).

survival of *P. palmivora* equal to 0.

*palmivora*

0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0

Scores of leaf susceptibility

control

IFC5, P7 and SCA6.

a ab

B104

B132

ab abab

<sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup>

B84

B127

B85

ab ab ab

<sup>a</sup> <sup>a</sup>

B122

B108

B92

ab ab

a <sup>a</sup> a

B103

B54

B14

ab ab ab ab

a

B

B

B141

Fig. 3. Effect of the bacterial strains on the leaf discs susceptibility to *P. palmivora* for clones

With the moderately resistant clone (P7), the rating scores varied in the same proportions. In both cases, the analysis of variance revealed a significant (P<0.05) effect of bacteria and the Student Newman & Keuls test revealed 5 homogeneous groups of bacteria (a, ab, b, bc, and c). The best results were obtained with bacteria BI05 and B116 (group c). With the susceptible clone IFC 5, we note that the reduction of the rating scores of leaf susceptibility is relatively low. The scores varied from 3.8 to 2.8. The statistical analyses did not reveal any

Similarly, the effect of 57 *Trichoderma* isolates on *P. palmivora* was evaluated using leaf disk of three cocoa clones. For the susceptible clone (IFC5), the scores of leaf susceptibility varied from 2.4 to 0.02 respectively with the isolates T39 of *T. spirale* and T55 of *T. virens*. With the moderately resistant clone (P7), we note a reduction of the scores, which varied from 2.04 to 0.03 respectively with the isolates T39 of *T. spirale* and T28 of *T. virens*. With the resistant

Strains of bacteria

B135

B106

b

a a <sup>a</sup> <sup>a</sup>

b

ab

a

B99

B86

<sup>b</sup> b b

a a a

B113

B140

B88

B

B10

B20

B129

<sup>b</sup> b b bc bc bc bc

a a a

a a

B

B11

B10

B102

<sup>b</sup> bc bc c c

a

B123

B114

<sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup>

B116

B105

4

significant (P>0.05) differences between the treatments and the untreated control.

clone (SCA6), the scores were less than 1.5 for all the isolates of *Trichoderma* (Fig. 4).

Fig. 4. Effect of *Trichoderma* strains on the leaf discs susceptibility to *P. palmivora* for clones IFC5, P7 and SCA6.

#### **3.3.3 Field efficacy of** *Trichoderma* **in the control of the black pod disease due to**  *P. palmivora*

The effects of 4 species of *Trichoderma*, applied to the cacao trees were compared. The results obtained are presented in Table 3. The final percentages of rotten pods for year 1, were 11.75, 8.44, 7.34, and 3.6 respectively with the isolates T4 of *T. spirale*, the isolate T40 of *T. harzianum*, the isolate T7 of *T. virens* and the isolate T54 of *T*. *asperellum*. These percentages were lower than 18.33 obtained with the untreated control, indicating a reduction of the yield losses from 36 to 80 %. For year 2, the final percentages of diseased pods were lower than that recorded in year 1. For both years of study, the calculated efficacy index was


Table 3. Reduction of the final losses due to the black pod disease and efficacy (%) of 4 species of *Trichoderma* during 2 years of field trials after the application

Isolation and Identification of Indigenous Microorganisms of Cocoa Farms in

*Phytophthora* sp, susceptible to be used in the control the black pod disease.

subjected to the attacks of *Rhizoctonia* sp., *Colletrotrichum* sp. and *Phytophthora* sp.

The effects of the isolates T4 of *T. spirale*, T7 of *T. virens* and T40 of *T. harzianum* in the field and the isolate T54 of *T. asperellum* on *P. palmivora*, showed a reduction of the incidence of the black pod disease due to *P. palmivora*. However, this effect is more striking on cacao trees treated with *T. asperellum*. Similar results were obtained by Tondje et *al*. (2007) in Cameroon after evaluating the potential effect of the isolates of *T. asperellum* on the incidence of the black pod disease due to *P. megakarya*. This reduction would be due to the mycoparasitic effect of *T. asperellum* against *Phytophthora* sp. Indeed, *T. asperellum* penetrates and destroys the propagules of *Phytophthora* on the flower cushions, thus reducing the quantity of inoculums of this parasite. Besides the mycoparasitic effect on the propagules (mycelium and sporocystes) of *P. palmivora*, the production of cellulase by *T. asperellum* in the presence of *Phytophthora* sp. would also be determining. The synergy of the modes of action in the

Côte d'Ivoire and Assessment of Their Antagonistic Effects Vis-À-Vis *Phytophthora palmivora…* 313

microorganisms. An analysis of the list of the collected microorganisms revealed the existence of fungi and bacteria identified by several authors as having antagonistic effect against pathogens responsible for plant diseases. This is the case of the genus *Trichoderma* in which several species have been tested for the control of cocoa diseases (Sanogo *et al.,* 2002; Krauss and Soberanis, 2001; Tondje *et al.,* 2007; Samuels, 1996). In the group of the bacteria, the antagonistic effect of the genus *Bacillus* was demonstrated by Shari Fuddin, 2000. The direct confrontation tests carried out *in vitro* between *P. palmivora* and the *Trichoderma* isolates revealed an antagonistic effect going from the inhibition of mycelia growth (fungistatic effect), to the degradation and the disappearance of the mycelium of *P. palmivora* (mycoparasitic and fungicidal effect). Similar results were obtained with *Trichoderma harzianum* on *Fusarium oxysporum* (Hibar *et al.*, 2005). In Central America, similar results were also obtained with *Trichoderma stromaticum* in the control of witch's brooms disease of cacao and with *Trichoderma virens* against the black pod disease (Krauss and Soberanis, 2002). This study thus allowed to isolate and to purify several potential antagonists of

The evaluation of the effect of the bacterial strains on *P. palmivora* using the leaf disk test revealed a reduction of the leaf susceptibility to *P. palmivora* for resistant clones (SCA 6) and moderately resistant clones (P 7). On the other hand, this effect is less perceptible with the sensitive clone (IFC5). Similar results were obtained by Maurhofer et *al*. (1994) on tobacco, Duijff et *al*. (1997) on tomato and Chen et *al*. (1998) on cucumber. These results could reveal an increase in the level of the intrinsic resistance of the plant by the bacterial strains. The highest effects were obtained with bacterial strains B 105 and B 116, which makes them potential candidates for the biological control against *P. palmivora*. These two bacteria belong to the genus *Bacillus* endowed with an ability to sporulate, suggesting a good ability of dissemination within the framework of a biological control program. Also, on leaf disks, the effect of *Trichoderma* revealed a reduction of the size and the frequency of the necrotic lesions due to *P. palmivora*. Similar results were obtained by Bowers *et al*. (2001b) on leaf disk with *Phytophthora megakarya*. This effect of reducing leaf susceptibility results from the germination of the spores of *Trichoderma,* on the underside of the leaves, which probably inhibits or hampers the germination of the zoospores of *Phytophthora*. This germination would stimulate the mechanisms of defense, and consequently would strengthen the resistance to the penetration and the dissemination of the parasite. Similar results were obtained by Bigirimana et *al*. (1997), Howell et *al*. (2000), Sid Ahmed et *al*., 2000 and Harman et *al*. (2004) on bean, cotton, hot pepper and corn inoculated by *T. virens* and *T. harzianum*,

higher than 60 % for the isolate T54 of *T. asperellum*. The analysis of the evolution of the epidemic curves in each treated plot reveals that the isolate T54 of *T. asperellum* and the isolate T7 of *T. virens* substantially reduced the losses due to the black pod disease. Furthermore, these isolates delayed the onset of the epidemic of the black pod disease compared to the untreated control (Fig. 5).

Fig. 5. Effect of the applications of biofungicide containing various species of *Trichoderma* on the evolution of black pod disease in the field.
