Evaluation of plant extracts for tomato late blight control in Cameroon

Abstract

Late blight caused by Phytophthora infestans is the most important disease of tomato in Cameroon. Three experiments are conducted to assess the fungitoxicity of methylene chloride/methanol leaf extracts of seven plants on P. infestans development. The extracts (3%) are evaluated for sporangial germination inhibition, latent and incubation periods, lesion size and late blight severity on tomato plants. Inoculated leaflets and plants are incubated in the greenhouse at 21°C during the day and 18°C during the night, with a 12 h photoperiod and for 7 days. Sporangial germination is significantly inhibited by Tephrosia vogelli and Entandrophragma angolense extracts, while disease progression is highly limited by Ageratum houstonianum, Tephrosia vogelli, Clausena anisata and Entandrophragma angolense extracts. The fungitoxic effects of some of the extracts is comparable to synthetic fungicides, metalaxyl and maneb, used as positive controls, indicating their potential as components of an integrated pathogen management programme for tomato late blight. Eucalyptus saligna, Deinbollia saligna and Garcinia smeathmannii extracts have low efficiency.

Keywords:

• fungitoxicity
• Phytophthora infestans
• plant-derived products
• tomato
• late blight

Introduction

Tomato (Lycopersicon esculentum Mill) is the most widely cultivated vegetable in Cameroon. The importance of this crop is due to its high nutritive value in both energy and protein content. However, continued tomato production in the highland agro-ecologies of Cameroon and elsewhere is consistently threatened by late blight, caused by the oomycete Phytophthora infestans Mont. de Bary. Fruit yield losses in Cameroon could reach to 100% due to late blight disease (Fontem et al. 2005).

The most common disease control measures involve frequent fungicide applications. However, late blight management with synthetic fungicides is usually limited because of a development of fungicide resistance by P. infestans. Reduced metalaxyl efficacy in late blight management has been linked to the intensive use of this fungicide in Cameroon and elsewhere (Fontem et al. 2005; Dorn et al. 2007). Furthermore, increasing concern for public health and the environment and expanding competition in the agricultural market motivates growers to seek disease control strategies that use reduced amounts of synthetic fungicides. For these reasons there is a need for new and effective means of disease control that pose less risk to human health and the environment.

Disease management using biopesticides such as plant-derived products and antagonistic microorganisms is an interesting alternative in late blight management (Regnault-Roger et al. 2005). Several studies have reported the effects of plant-derived products on P. infestans development (Stephan et al. 2005; Olanya & Larkin 2006; Dorn et al. 2007). Nevertheless, it is important to investigate efficient plant-derived products that are native to Cameroon to facilitate chances of their adaptation to local conditions and ease of exploitation. Many local plants in the region are reported to possess inhibitory properties in vitro against various pathogens (Fontem et al. 2006). However, most studies dealing with these compounds were limited to in vitro tests and little is known about the in vivo efficacy of the compounds against P. infestans. Consequently, this work was designed to evaluate the effect of seven plant extracts on P. infestans sporangial germination and on late blight development in tomato in vivo.

Materials and methods

Preparation of plant extracts

The leaves of the seven plants given below with their common names and families were collected in the western highlands of Cameroon during the months of June to October: Tephrosia vogelii Hook.f. (Fish poison, Fabaceae), Entandrophragma angolense Welw.C.DC. (Tiama, Meliaceae), Eucalyptus saligna Sm (Sydney bluegum, Myrtaceae), Clausena anisata Willd.Hook (Horsewood, Rutaceae), Garcinia smeathmannii Oliver (False chew-stick, Clusiaceae), Ageratum houstonianum P.Mill (Floss flower, Asteraceae) and Deinbollia saligna Keay (Dune soap berry, Sapindaceae).

Plant leaves were air dried (25–27°C) for 10 days, weighed to determine dry matter content, ground to obtain a fine powder and finally kept in plastic bags at ambient temperature until used for extraction. For each plant, a 500 g sample of fine powder was extracted at 27°C for one week by putting in a mixture of methylene chloride and methanol (1:1 vol/vol) at a ratio of 1:8 (wt/vol, dry powder/solvent). The extraction solvent was selected on the basis of previous experiments conducted using various solvents including aqueous extracts. The extract was filtered through two layers of cheesecloth and vacuum-dried at 40–45°C to obtain a paste. The extraction process yielded 8–16% paste for the various extracts. Extract solutions were prepared by dissolving 0.6 g of each paste in 20 mL of sterilized distilled water (SDW) containing 0.05% Tween 20 and a concentration of 3% was obtained.

Preparation of P. infestans sporangial suspension

Phytophthora infestans was isolated from infected tomato leaves obtained from the field as described by Fontem et al. (2005) and identified at the phytopathology laboratory of the University of Dschang, Cameroon. Cultures of a single isolate of P. infestans were grown on V8 medium amended with 150 ppm ampicillin and 100 ppm rifamycin, in 90 mm diameter Petri dishes for 10–14 days at 18°C in darkness. The inocula were collected with a fine brush into 10 mL SDW containing 0.01% Tween 20. The sporangial suspension was filtered through a double layer of cheesecloth to remove mycelial fragments and diluted to 2×10⁴ sporangia/mL with the aid of a haemocytometer. The inoculum was incubated at 8°C for 1 h to promote zoospore release prior to use.

Inhibition of sporangial germination by plant extracts

The effect of the plant extracts was evaluated in comparison to two synthetic fungicides—metalaxyl at 0.04% (added with cuprous oxide at 0.2%) and maneb at 0.53% —that are commonly used in tomato late blight management in Cameroon. According to primary studies examining the fungitoxicity effects of various concentrations on sporangial germination and mycelia growth of P. infestans (Fontem et al. 2006) and their phytotoxic effects on tomato and potato leaves, extract concentration of 3% was chosen in the study. Experiments were set in a completely randomized design with four replications (one Petri dish per replicate).

Mixtures (1:1, 20 µL of sporangial suspension of P. infestans and 20 µL of each extract and synthetic fungicides) were prepared and dropped on slides with wet Whatmann paper underneath. 20 µL SDW containing 0.05% Tween 20 and 20 µL spore suspension served as a control treatment. The Whatmann paper was then taken from the slide and placed in the centre of a 90 mm diameter plate containing 1.5% water agar medium. The Petri dishes were put in an incubator at 18°C in the dark. After 24 h, a fragment of water agar with the Whatmann paper carrying the sporangia on the upper side was transferred to a microscope slide, coloured with a drop of lactopherol and covered for microscopic examination. The number of germinated and non-germinated sporangia were counted on 200 sporangia/slide and the average germination rates of sporangia with extract (Ge) and without extract (Gc) determined. Percent germination inhibition (%I) was calculated according to the formula:

The experiment was conducted four times.

Aggressiveness of P. infestans on detached tomato leaflets treated with plant extracts

Seeds of tomato cultivar ‘Roma VF’ obtained from a local shop in Dschang served to produce sensitive host plants for P. infestans. Fully mature healthy leaflets were detached from the middle canopy layer of plants (7 to 8 weeks old) raised in a greenhouse. The detached leaflets were washed with SDW and the base of each leaflet was covered with a piece of moist cotton to reduce leaf desiccation. Single leaflets were then placed upside down (abaxial surface up) in 90 mm diameter Petri dishes containing moistened filter paper to form a humidity chamber. The experimental design was completely randomized with four replicates (four Petri dishes per replicate).

Extracts at 3% and synthetic fungicides were applied on detached leaflets by means of a small paint brush. After the leaflets had dried (1 h) they were then inoculated with a single drop (50 µL, 2×10⁴ spores/mL) of sporangial suspension applied to the midrib of the leaflet using a Pasteur pipette. Control treatments contained a drop of SDW containing 0.05% Tween 20 and were also inoculated. Untreated and non-inoculated leaflets served as blank controls. The Petri dishes were kept in the greenhouse at 21°C during the day and 18°C during the night, with 12 h photoperiod. After 7 days of incubation, the length (L) and width (l) of the lesion were measured for each leaflet and lesion size (S in cm²) was calculated using the formula:

The experiment was repeated three times.

Late blight latency and severity on whole tomato plants treated with plant extracts

Forty-day-old tomato plants raised in the greenhouse and thinned to one plant per pot were used for the experiment. Treatments were applied by spraying the extract solution (3% concentration) on the tomato foliage to run-off, using a handheld sprayer. At about 1 h after treatment when the leaves had dried, the inoculum suspension was sprayed on the leaves (1 mL/leaf) using a handheld sprayer. Control treatments included plants inoculated after treatments with metalaxyl, maneb and SDW containing 0.05% Tween 20. Untreated and non-inoculated plants served as blank controls. After inoculation, plants were allowed to dry, then covered with a polyethylene bag for 24 h to maintain the high humidity atmosphere around the foliage and immediately placed in the greenhouse at 21°C during the day and 18°C during the night, with 12 h photoperiod for 7 days. The experiment was conducted in a complete randomized block design with four replicates and four plants/replicate, and then repeated three times.

The plants were observed daily for first symptoms (incubation period) and first sporulation (latent period). After the appearance of symptoms, late blight severity (the proportion of leaf area infected) was estimated visually on each plant with the aid of the modified Horsfall-Barrat rating scale of 1 to 12 (1 = 0%, 12 = 100% disease severity) (Berger 1980).

Statistical data analysis

All statistical analyses were performed using SPSS 15.0. Data were transformed using the arcsine square root (x/100) transformation for percentage sporangial inhibition and disease severity, and square root (x + 0.5) transformation for lesion size. The data were subjected to an analysis of variance (ANOVA) and treatment means for each factor (spore germination, aggressiveness on detached leaves, latent and incubation periods, disease severity) were separated using Tukey's honest significant difference (HSD) multiple comparison tests at P < 0.05. Data are reported as means of four experiments with four replicates per experiment±standard error of mean (SEM). Simple Pearson's correlation coefficients (r) were calculated for the relationship between the five factors assayed.

Results and discussion

Inhibition of sporangial germination by plant extracts

T. vogelii extract showed the strongest inhibition of sporangial germination of P. infestans (67.95%) among plant extracts, followed by E. angolense extract (53.21%) (Table 1). The result obtained with T. vogelii extract is similar to that obtained in vitro on the hyphal growth of the same pathogen (Fontem et al. 2006). There was no significant difference (P < 0.05) observed between the inhibitory effects of T. vogelii extract and that of the synthetic fungicide maneb. E. saligna, C. anisata, A. houstonianum, G. smeathmannii and D. saligna extracts were less effective in inhibiting sporangial germination.

Table 1  Effect of plant extracts at 3% on four distinct stages of Phytophthora infestans pathogenesis: percent sporangial germination inhibition after 24 h of incubation on 1.5% water agar medium at 18°C in darkness, lesion size of late blight on 7–8-week-old detached tomato leaflets in Petri dishes, incubation period and percent severity of late blight on tomato plants incubated in the greenhouse for 7 days at 21°C during the day and 18°C during the night, with 12 h photoperiod

Treatment	Sporangia inhibition (%)	Lesion size (cm^2)	Incubation period (days)	Disease severity (%)
Tephrosia vogelii	67.95±1.76 c^*	2.18±0.46 fg	4 a	8.09±0.71 e
Entandrophragma angolense	53.21±2.06 b	3.68±0.36 e	3 b	8.10±0.73 e
Eucalyptus saligna	31.45±1.10 e	9.80±0.80 d	3 b	20.98±1.98 d
Clausena anisata	20.63±1.25 f	3.00±0.23 ef	5 a	4.73±0.91 f
Garcinia smeathmannii	12.83±0.53 g	19.80±0.17 c	2 c	48.05±1.55 b
Ageratum houstonianum	14.88±1.06 g	0.18±0.17 hi	3 b	4.20±0.41 f
Deinbollia saligna	6.50±1.29 h	20.50±0.58 b	1 d	28.75±0.96 c
Metalaxyl	72.45±2.80 a	0.40±0.49 hi	–^†	0.00±0.00 g
Maneb	70.33±2.05 bc	1.33±0.98 gh	5 a	3.20±0.36 f
Uninoculated control		0.00±0.00 i	–^†	0.00±0.00 g
Distilled water-sprayed		23.08±1.43 a	1 cd	55.50±1.73 a
^*Means (±SEM) in a column with the same letter are not significantly (P < 0.05) different according to Tukey's HSD multiple comparison test. Values are means of four experiments with four replicates per experiment
^†No late blight lesion developed during the 7-day observation period

Aggressiveness of P. infestans on detached tomato leaflets treated with plant extracts

Few late blight lesions developed on leaflets treated with A. houstonianum extract (0.18 cm²), metalaxyl (0.4 cm²) and maneb (1.33 cm²) (Table 1). The inhibitory effects of T. vogelii (2.18 cm²), C. anisata (3 cm²) and E. angolense (3.68 cm²) extracts were comparable to that of maneb. Other extracts produced less inhibitory effects, although all the extracts tested reduced lesion size compared with control plants sprayed with water (23.08 cm²).

Disease latency as affected by treatment with plant extracts

In the greenhouse, non-treated tomato plants sprayed with water and plants treated with D. saligna produced first late blight symptoms in one day. The latent period was longer on the other extract-treated plants (Table 1). No late blight symptoms were observed during 7 days of incubation on plants treated with metalaxyl and non-inoculated plants. Generally, spores could be observed on the lesions only 5 to 10 h after the appearance of the first symptoms. Consequently, there were no significant differences between incubation and latent periods. It should be remembered that lesions can be observed within minutes after infection with some isolates of P. infestans.

Late blight severity on whole tomato plants treated with plant extracts

Four plant extracts had preservative effects on late blight development on tomato plants in the greenhouse after 7 days: A. houstonianum (4.20%); C. anisata (4.73%); T. vogelii (8.09%); and E. angolense (8.10%) extracts (Table 1). Although A. houstonianum and C. anisata extracts did not have promising antifungal properties in vitro, the protection they afforded plants against infection by reducing disease severity was strong enough. The possible reason for this discrepancy may be due to the activation of plant defence responses by the two extracts. Plant-derived products and antagonistic microorganisms with a similar mode of action have been reported (Godfrey et al. 2000; Regnault-Roger et al. 2005; Dorn et al. 2007). Microscopical observations and characterization of plant cell responses under light microscope are currently under investigation to elucidate it. The least effective treatment was G. smeathmannii extract followed by D. saligna and E. saligna extracts.

Disease suppression on detached leaflets was generally associated with disease suppression on whole tomato plants (r=0.931 with the correlation significant at the 0.01 level) and negatively correlated with disease latency (r=-0.717 with significance at the 0.05 level). There was a moderate significant correlation at the 0.05 level between the incubation period and P. infestans sporangial germination and late blight severity on entire tomato plants, with r values of 0.697 and -0.680, respectively. The linear correlation coefficient between the percentage inhibition of sporangial germination and late blight severity on detached leaflets (r=-0.652) and entire tomato plants (r=-0.602) was, however, negative and non-significant. Overall, T. vogelii and E. angolense extracts were the most effective in affecting the different stages of P. infestans pathogenesis. This study indicates that there is potential for the use of plant extracts to inhibit P. infestans development. Biopesticides alone or as mixtures may be exploited as part of an integrated pathogen management strategy for tomato late blight disease.

Acknowledgements

This work was funded by a grant from the Agence Universitaire de la Francophonie. We thank Salomon Pangui for his assistance in the laboratory and James Aipa for useful comments.