Antifungal Effect of Cymbopogon citratus, Eucalyptus camaldulensis and Azadirachta indica Oil Extracts on Sorghum Seed-Borne Fungi

MATERIALS AND METHODS

Fungal isolates: Isolates of Colletotrichum graminicola, Fusarium moniliforme and Phoma sorghina were recovered, in 2003 at DSHC (former DGISP), Denmark, from sorghum seeds collected in 2002 in Burkina Faso. The isolates were preserved on dry sterile soil and sand mixture in glass tubes for long term storage. A dense suspension (2-5 mL) of mycelium plus spores scraped from agar cultures was added into each vial and mixed with the soil. The screw caps were wrapped with Parafilm^® to prevent dehydration. The tubes were stored at 5°C until use.

Sorghum seed samples: Naturally infected sorghum seeds from farmers own saved seed lots were chosen based on the pericarp colour (red and white) and on the incidence of C. graminicola, P. sorghina and F. moniliforme revealed by the standard blotter test performed at DSHC according to Mathur and Kongsdal (2003) (Table 1).

Botanicals and synthetic fungicide: Essential oils and crude oil were used in this study (Table 2). Essential oil (CEO) of lemongrass (Cymbopogon citratus), essential oil (EEO) of Eucalyptus (Eucalyptus camaldulensis) and crude oil (NCO) of neem (Azadirachta indica) were tested. The essential oils were prepared by the Research Institute for Applied Sciences and Technology (IRSAT) of Burkina Faso. Extraction was performed by using stream distillation. Throughout the study, the commercial fungicide Dithane M-45 (Zinc-manganese ethylene bisdithiocarbamate) (Dow chemicals) was used as positive control.

Testing effect of oil extracts on fungal growth: One isolate of each test fungal species was tested in three replicates for each concentration of Essential Oil (EO) using the agar diffusion method. Four concentrations of essential oils (2, 4, 6 and 8%) were tested. The oils were emulsified using 0.1% sterilized water agar (Remmal et al., 1993). Conidial suspensions of 10⁶ conidia mL^-1 were prepared from 7 days agar cultures. A drop of 50 μL of the suspension was streak on agar plates (9 cm diameter) containing 25 mL PDA. Ten microliters of the EO emulsion were loaded into each of the four wells (2 mm diameter) made in the agar medium. Control plates were loaded with either water agar (0.1%) or Dithane (0.3%,w/v). The plates were incubated at 25±1°C under cycles of 12 h NUV light/12 h darkness for 2-3 days. The Inhibition Zone (IZ) around each well was evaluated by measuring two perpendicular diameters for each well. The mean of diameters of the four wells gave the IZ for each replicate.

Seed treatment with oil extracts: The seeds were treated with an emulsion of essential oils (Eos) in 0.1% water agar solution.

Table 1:	Sorghum seed samples collected in south western region of Burkina Faso and their natural infection rates by pathogenic fungi

Table 2:	Plant species collected from Ouagadougou, Burkina Faso environment and tested in this study

Four concentrations were considered based on preliminary test (data not shown). The EO emulsions were tested at the concentrations 2, 4, 6 and 8%. One hundred microliters of the mixture were applied per gram of seeds in 50 mL-Erlenmeyer flasks according to Adegoke and Odesola (1996). Negative controls were treatment with water agar and untreated seeds. The positive control was treatment with Dithane M-45 at the rate of 0.3% of seed weight. After treatment, the flasks were covered with Parafilm^®, shaken for 5 min and maintained overnight at 20-22°C in darkness.

Testing effect of oil extracts on seed health: Two hundred seeds were tested for each treatment. The oil-treated seeds were plated in Petri dishes containing three layers of moistened blotter papers according to Mathur and Kongsdal (2003). A set of four plates (100 seeds) was considered as replicate. Petri plates containing 25 seeds were considered as sub-replicates and randomly arranged in the incubation room. The positive control was treated with Dithane M-45 (0.3%, w/w). The negative control was treated with water agar. Set of untreated seeds was also considered. The disease incidence was recorded as percent of seed bearing a given fungus per replicate.

Testing effect of oil extracts on seedling emergence and growth: Two hundred seeds per treatment (100 seeds per replicate) were used for the growing-on test. Seeds were treated as described for the seed health experiment and were planted in standard peat soil (Weibull K-Soil) in plastic pots (14x13x6 cm), i.e., 25 seeds per pot and eight pots per treatment. After sowing, the pots were incubated in growth chamber at 28±2°C under cycles of 12 h fluorescent light/12 h darkness. Four pots per replicate were randomly arranged in the trays. After 3, 7 and 14 days, seedling emergence was evaluated and percentage of emerging seedlings calculated. After 14 days of incubation, the seedlings from each replicate were cut at the soil level and weighed.

Testing effect of oil extracts on fungal seed to seedling transmission: After weighing, 20 fresh plants randomly picked per replicate (i.e., five plants per pot) were assayed for recovery of the four seed-borne fungi cited above. The first leaves were removed and plated on moistened blotters. After surface disinfection and sterilization in 70% ethanol for 30 sec and in 1% sodium hypochloride for 1 min, respectively, stems were cut aseptically into smaller sections from the base up to ca 10 mm. These segments were plated on three layers of moistened blotter papers in Petri dishes and incubated at 25±1°C under NUV light (12 h/12 cycles). Stem and corresponding first leaf were plated and marked accordingly. After five days, the fragments were inspected under stereomicroscope for the presence or absence of the target fungi.

Data analyses: Treatment effects were determined by one-way analysis of variance using a completely randomised design. The significance of differences between treatments was determined, using the Least Significant Difference (LSD) test of Statgraphic statistical software, version 5. Pearson correlation was also performed to reveal the relationships between different parameters.

RESULTS

Effect of oil extracts on fungal radial growth: Isolates of C. graminicola, P. sorghina and F. moniliforme recovered from naturally infected seeds were tested in vitro. The results of this study showed differences in sensitivity of the different fungal species and in potency of essential oils. C. citratus essential oil exhibited antifungal activity against all the three species of fungi. E. camaldulensis essential oil showed very low level of antifungal activity and neem crude oil had no adverse effect on the growth of the test isolates (Table 3). C. graminicola was highly sensitive to essential oil of C. citratus; 76.2% inhibition was obtained using 8% emulsion. Fifty percent growth inhibition was recorded for P. sorghina at the highest concentration, while F. moniliforme was less sensitive to the lemongrass essential oil (21.4-41.3%). A dose dependent inhibition of C. graminicola mycelial growth was caused by lemongrass essential oil.

Effect of oil extracts on seed health: Statistical analyses showed significant effects of treatments on C. graminicola recorded on sample 47056 and on P. sorghina and F. moniliforme evaluated on sample 205/214. C. citratus essential oil was more effective than that of E. camaldulensis on C. graminicola (Table 4). The highest percent reduction of C. graminicola (94.4%) was recorded at the concentration 6%, while only 16.7% reduction was obtained with E. camaldulensis oil at concentration 8%. Essential oil from C. citratus was very effective against P. sorghina with reduction of infection percent ranging from 71.7-95.1%, while infection by F. moniliforme was reduced to levels ranging from 20.7-62.1% on sample 205/214. Both P. sorghina and F. moniliforme were less sensitive to E. camaldulensis essential oil (13.6 and 44.8% reduction of infection, respectively).

Table 3:	Effect of oil extracts on mycelial growth inhibition of Colletotrichum graminicola, Phoma sorghina and Fusarium moniliforme1
1: Data are mean diameters (mm) of inhibitory zones round 12 wells per concentration, 2: Azadirachta indica crude oil, essential oils of Cymbopogon citratus and Eucalyptus camaldulensis tested at different concentrations (v/v, oil/water agar). Mean values in the same column followed by the same letter are not significantly different. Data within brackets are the percent inhibition compared to treatment with Dithane M-45

Table 4:	Effect of seed treatment with oil extracts on the incidence of Colletotrichum graminicola, Phoma sorghina and Fusarium moniliforme1
1: Data are mean N. of infected seeds per replicate, 2: Essential oils tested at four different concentrations (v/v), 3: C.g. = C. graminicola, P. s. = P. sorghina, F. m. = F. moniliforme; Data within brackets are the percent reduction compared to negative control (WA); WA: water agar, DIT: Dithane-M45 used at dosage 0.3% (w/w), NT: non-treated, nt : not tested. Mean values followed by the same letter are not significantly different (p<0.05)

Effect of oil extracts on fungal seed to seedling transmission: Treatment effects on transmission of the target fungi from seeds to leaves and stems were not statistically significant. Therefore, Fig. 1 describe the trend of the seedling infection compared to percent infection of seed. The transmission rate was higher than expected, based on the results from the seed health testing. On sample 47056, having the highest initial incidence of C. graminicola (10.5%), leaf and stem transmissions were observed with percent infection more than 10% (Fig. 1a). C. citratus essential oil reduced interestingly the transmission of C. graminicola and P. sorghina, as compared to the water agar control (Fig. 1a, b). E. camaldulensis had no adverse effect on the transmission of the target fungi (Fig. 1). As shown in Fig. 1b, P. sorghina was less transmitted to both leaves and stems, while F. moniliforme appeared highly transmitted to seedlings of both samples tested (Fig. 1c, d). The synthetic fungicide Dithane M-45 also appeared less effective against F. moniliforme on sample 205/214 (Fig. 1d). On sample 205/214 exhibiting 20.25% seed infection in the original sample, there was no apparent difference in seed-to-seedling transmission of F. moniliforme after treatments.

Effect of oil extracts on seedling emergence: A highly significant treatment effect on seedling emergence was found at the different dates of evaluation (Fig. 2). All the treatments were compared to the water agar control for both seed samples (47056 and 205/214) displaying potential emergence of 84 and 82%, respectively. Neem crude oil showed high inhibitory effect on seed germination and emergence on both samples. Emergence of 40% was hardly obtained with neem oil across the sample and the dates of evaluation (Fig. 2). Essential oil from C. citratus showed a clear depressive effect on sample 47056 when high concentrations were used (Fig. 2a). Lower concentrations (2 and 4%) were as good as the negative control (ca 100% emergence). Emergence was 10% higher with Dithane treatment than that of the negative control (Fig. 2a). The same trend was observed with sample 205/214 Fig. 2c). The highest concentration of C. citratus essential oil (8%) was markedly harmful to emergence. Interestingly, concentration of 6% was as effective as 2 and 4% in having no depressive effect on seedling growth. In return, essential oil from E. camaldulensis was slightly more effective than the control with percent emergence ranging from 100-110% on the white sorghum sample 47056 (Fig. 2b).

Fig. 1:	Effect of essential oils on seed infection and seed to seedling transmission of C. graminicola, on sample 47056 (a), P. sorghina on sample 205/214 (b) F. moniliforme on samples 47056 and 205/214, respectively (c, d)

Fig. 2:	Effect of treatment with essential oils of Cymbopogon citratus (Cc) (a, c), Eucalyptus camaldulensis (Ec) (b, d) at four concentrations and neem crude oil on the emergence percentage of two sorghum samples (47056 and 205/214) compared to water agar control (potential emergence of the samples 47056 and 205/214 are 84 and 82%, respectively)

Similarly, on the red sorghum sample 205/214, the effects of the different concentrations used were comparable to the controls. On both samples, concentrations of E. camaldulensis essential oils had no adverse effect on seedling emergence which percentages ranged from 95-110% compared to the negative control (Fig. 2b, d).

Table 5:	Effect of plant extracts on sorghum seedling growth rate (nb day-1) and total biomass (g)2
1: Data are mean numbers of seedlings per replicate; 2: Data are mean fresh weights of seedlings per replicate; 3: A.. indica crude oil, essential oils of Cymbopogon citratus and Eucalyptus camaldulensis tested at different concentrations (v/v). Mean values in the same column followed by the same letter are not significantly different (p<0.05)

Table 6:	Relationships between fungal radial growth inhibition seed health and seed to seedling transmission of Colletotrichum graminicola (a), Phoma sorghina (b) and Fusarium moniliforme (c) after treatment with essential oils
1: Inhibition zones round the wells containing the antagonist compound tested. Values are Pearson product moment correlation coefficients followed by *: p<0.05, significant, **: p<0.01, highly significant, NS: Non significant

Effect of oil extracts on seedling growth rate and biomass production: Neem oil and both essential oils significantly affected seedling growth rate and total biomass (Table 5). Neem crude oil exhibited the greatest inhibitory effect on growth rate (3.6 and 4.5 seedlings day^-1 for samples 47056 and 205/214, respectively and biomass (5.6 and 7.0 g for samples 47056 and 205/214, respectively. C. citratus essential oil at concentration of 8% significantly reduced both growth rate and total biomass, whereas E. camaldulensis essential oil had no depressive effect (Table 5).

Relationships between inhibition of fungal radial growth, seed health and seed transmission: Since essential oils showed effects on different parameters, the influence between those parameters was evaluated (Table 6). The inhibitory effect of essential oil treatments on fungal radial growth was highly and negatively correlated to the incidence of target fungi on seed; correlation coefficients ranging from -0.76 to -0.93. The transmission of C. graminicola from seed to stems was weakly and positively correlated to seed infection (0.59). No significant correlation was found between seed infection of P. sorghina and its seed to seedling transmission. There were positive and low relationships between stem and leaf transmission of C. graminicola and P. sorghina. Mycelial growth inhibition in vitro and seed to stem transmission of F. moniliforme were negatively and highly correlated (-0.86), while weak and positive correlation was found between seed infection and stem transmission (0.68).

DISCUSSION

Phoma sorghina and Fusarium moniliforme are responsible of grain mould on sorghum grown in Burkina Faso. Furthermore, Colletotrichum graminicola is the most threatening grain, leaf and stalk disease of sorghum in smallholder farmers fields. These fungi were the main pathogens encountered in many seed samples from Burkina Faso analysed at the Danish Government Institute of Seed Pathology for Developing Countries, Denmark (Somda et al., unpublished data). Extracts from local plants (botanicals) may provide resource-poor farmers with an option to control seed-borne pathogens of their own saved seeds, using locally available, environmentally friendly methods. The overall aim sought in this study was to evaluate the efficacy of essential and crude oils in controlling major facultative pathogens carried by sorghum seeds without inhibiting seed germination and seedling growth. The agar diffusion test showed that essential oil emulsions exhibit an inhibitory property against C. graminicola, P. sorghina and F. moniliforme. Neem crude oil had no effect on the target fungi. This contrasts with the results of Amadioha (2000), who found radial growth reduction of Pyricularia oryzae Cav. by neem oil extracts. The lack of inhibitory activity may be a result of low concentration or lack of active constituents, or simply because of the oil extraction method. Nevertheless, results of the radial growth experiments showed that this modified agar diffusion method was efficient in screening low volumes of essential oils. C. citratus essential oil showed the highest antifungal activity against C. graminicola, P. sorghina and F. moniliforme, as compared to the fungicide Dithane M-45. E. camaldulensis essential oils were slightly effective at high concentrations. These results showed that the inhibitory effect of C. citratus essential oil was concentration dependent. Interestingly, strong relations were obtained between the inhibition of fungal radial growth by essential oils and their effects on seed infection along with seedling growth regulation. This result infers that the agar diffusion test can actually be used as a first step when evaluating the potency of plant extracts to control seed-borne pathogenic organisms. Another important point raised by present study is that essential oils were effective in controlling the test fungi on sorghum seeds. Of the three plants extracts evaluated, C. citratus essential oil was the most potent. The concentrations of 6 and 8% caused the greatest reduction of seed infection at levels comparable to the fungicide Dithane M-45. E. camaldulensis essential oils were inferior as fongitoxicant on sorghum seeds, confirming its lack of potency demonstrated in vitro. Studies on the uses of plant extracts to control grain spoilage and phytopathogenic fungi are well documented (Dubey et al., 2000; Bajwa et al., 2003; Aladi et al., 2005; Yulia et al., 2006; Cardenas-Ortega et al., 2007). The fungitoxic effects of lemongrass essential oil were in agreement with the results obtained by Banjole and Joda (2004), Nguefack et al. (2004) and Abd-El-Khair and Wafaa (2007). As seed treatment, lemongrass essential oil at concentrations 6 and 8% was more effective against C. graminicola in reducing seed infection by 66.7 to 94.4% in the white sample (47056) and by 71.7 to 95.1% in the red sorghum sample 205/214. More than 50% of the growth of F. moniliforme was reduced by C. citratus essential oil both in vitro and in vivo on seed whereas E. camaldulensis essential oil was less efficient even at high concentrations. The weaker control effect obtained with E. camaldulensis essential oil against all tested fungi could be attributed to its contents of known antimicrobial compounds. C. citratus from Burkina Faso contained citral a and b (Menut et al., 2000), which is absent in E. camaldulensis essential oils (Maximous, 2004).

The potential use of plant oil extracts for the control of plant diseases requires identification of extracts which inhibit the growth of pathogens at non-phytotoxic concentrations. C. citratus essential oil was efficient in reducing seedling infection of C. graminicola, P. sorghina and, in some instances, F. moniliforme at levels comparable to the fungicide Dithane M-45. Low concentrations (2 and 4%) of C. citratus essential oil improved seedling emergence, while high concentrations maintained seedling emergence at a level equivalent to the potential emergence of the original sorghum seed samples.

CONCLUSION

The present study concludes that lemongrass essential oil at 6% concentration can be effectively used to treat sorghum seed against pathogenic fungi, namely C. graminicola and P. sorghina. These findings prompt further investigations on the effectiveness of this oil extract under field conditions.

ACKNOWLEDGMENTS

The first author wishes to thank DANIDA and the Danish Seed Health Centre for Developing Countries (DSHC), formerly DGISP, for providing the grant and opportunity for this work. Authors are also grateful to Ms Henriette West for reviewing the manuscript.