Ridding Phytophthora palmivora cultures of contamination by Colletotrichum gloeosporioides

0, 75, 100, 125 and 150 ppm a.i. concentrations of benomyl in Green Cocoa Mucilage Agar (GCMA) plates were evaluated for suppression of Colletotrichum gloeosporioides in Phytophthora palmivora plates contaminated by C. gloeosporioides. To do this, GCMA plates amended with the above concentrations of benomyl were seeded with mycelia plugs from a contaminated P. palmivora culture and radial growth rates of the resulting P. palmivora colonies were recorded. Mycelial bits from the centres, middle and peripheries of such colonies were examined at 7 days interval for P. palmivora and the contaminating C. gloeosporioides. Growth rates of pure C. gloeosporioides cultures were also studied on benomyl plates. Growth rates of 7.15, 3.10, 2.30, 2.05 and 1.60 mm/day were obtained for P. palmivora on plates, respectively, containing 0, 75, 100, 125 and 150 ppm benomyl. Growth rates of 2.95, 1.65, 1.60, 1.60 and 1.55 mm/day were respectively, obtained for the pure cultures of C. gloeosporioides indicating that on contaminated plates, growth of C. gloeosporioides will lag behind that of P. palmivora. On plates with 0 to 125 ppm benomyl, P. palmivora was detected at the centres, middle and peripheries of all colonies within the first week but C. gloeosporioides was restricted to the centres and middle portions of such colonies due to its slower growth rate. When subcultures were made at day 7 from the peripheries of colonies on 0 to 125 ppm benomyl plates onto benomyl-free plates, C. gloesporioides was not detected from the resulting cultures which were clean.


INTRODUCTION
The causal agents of black pod disease of cocoa in Ghana are Phytophthora palmivora, (Brassier and Griffin, 1979) and Phytophthora megakarya (Dakwa, 1987).The disease is a major constraint to cocoa production and causes severe economic losses worldwide.Van der Vossen (1999) reported that black pod disease causes about 44% of crop loss globally.Total yield losses in Ghana due to P. palmivora and P. megakarya are 4.9 to 19 % (Dakwa, 1984) and 60 to 100% (Dakwa, 1987), respectively.
Contamination of P. palmivora cultures by other fungi, notably Colletotrichum gloeosporioides is problematic.C. glooesporioides is a cosmopolitan saprophyte which invades tissues (Uchida and Kodaaka, 1997) and has been reported on diseased cocoa pods (Lass, 1985).Akrofi (personal communication, 2005) at the Cocoa Research Institute of Ghana (CRIG), observed that C. gloeosporioides was involved in severe chellere and pod rot disease of cocoa in Ghana.Thus, in culturing P. palmivora, the possibility of contamination by C. gloeosporioides is high.Such contaminated cultures are unsuitable for pathological and mycological studies on P. palmivora.Means of ridding such cultures of the contaminating C. gloeosporioides based on the use of a selective medium which selects for P. palmivora must be developed.Selective isolation of Phytophthora spp.by incurporating chemicals into the culture medium is well known.Ritchie (2002) indicated that benomyl can be used in a medium to culture fungi in the Phylum Oomycota, which includes Phytophthora palmivora (Alexopolous et al., 1996).Benomyl (50 % WP), a benzimidazole fungicide, is reported to suppress growth of a large number of fungi including many Deuteromycetes (Agrios, 1997) to which C. gloeosporioides belongs (Alexopolous et al., 1996).
One possible means we thought could be effective in cleaning P. palmivora cultures contaminated by C. gloeosporioides was by incorporating benomyl in the culture medium to selectively inhibit C. gloeosporioides.There is, however, no report on the concentration of benomyl that will effectively inhibit C. gloeosporioides while enabling growth of P. palmivora.
The main objective of the present study was to find means of cleaning P. palmivora cultures contaminated by C. gloeosporioides.Specific objectives, therefore, were to: 1. Determine the effect of different concentrations of benomyl on the growth of C. gloeosporioides and P. palmivora and 2. Determine the threshold period for subculturing from different portions of a contaminated culture of P. palmivora.

Preparation of selective medium for P. palmivora
Green Cocoa Mucilage Agar (GCMA) was prepared from the mucilage of cocoa beans according to the method of Awuah and Frimpong (2002).Fresh beans (250 g) from matured green cocoa pods was stirred vigorously with an electric stirrer for 10 min in 500 ml sterile distilled water (SDW) and the suspension was filtered through a double layer cheesecloth.Sodium hydroxide solution (0.1 M) was added to the filtrate (10% v/v) and agar added at 2% (w/v).The mixture was dispensed into 200 ml Erlmeryer flask (100 ml per flask).Benomyl (benlate 50% WP) was added to the content of the flasks to establish concentrations of 75, 100, 125 and 150 ppm a.i.(corresponding to 150, 200, 250 and 300 mg, respectively, of the benomyl powder).The media were autoclaved, dispensed into 9 cm diameter Petri dishes and used after 3 days.Medium without benomyl served as control.

Assay of media for selectivity against C. gloeosporioides
A 2-week-old culture of P. palmivora contaminated by C. gloeosporioides and growing on Oat Meal Agar (OMA) was the source of the two fungi.The original culture was obtained from the Cocoa Research Institute of Ghana (CRIG), Akim Tafo and named in the present study as CRIG-2.Subcultures of CRIG-2 (also contaminated by C. gloeosporioides) were maintained on GCMA before use for an experiment.Three replicate plates containing a particular concentration of benomyl were centrally seeded with 7 mm diameter mycelial plugs of CRIG-2 (one plug, top down, per plate) and incubated in the dark at 27 ± 2°C.Diameters of the resulting contaminated P. palmivora colonies were measured at 24 h interval for 5 days along two perpendicular planes and the mean diameter for each plate was calculated.From these, the average radial growth rate per day was determined.Additionally, mycelia bits were taken at 7, 14 and 21 days after incubation from the centre (0 to 15 mm), middle (15 to 30 mm) and periphery (outer 2 mm) of the contaminated P. palmivora cultures and were examined with a microscope for the presence or absence of P. palmivora and C. gloeosporioides.Because C. gloeosporioides was not usually detected at the peripheries of colonies, mycelia bits from the peripheries of all plates were plated on benomyl-free GCMA plates, grown in the dark and examined weekly (for 3 wks) as before to confirm absence of C. gloeosporioides.A set-up using a pure culture of C. gloeosporioides was maintained and radial growth rate of the fungus was determined as with the Phytophthora colonies.

Data analysis
Radial growth rate data were analysed with the analysis of variance (ANOVA) to assess the effect of benomyl concentrations on the growth of P. palmivora and C. gloeosporioides.These were compared with the least significant different (LSD) test at 5% probability level.

RESULTS
Radial growth rates of P. palmivora cultures (in contaminated states) and those of pure cultures of C. gloeosporioides on plates amended with different concentrations of benomyl are presented in Table 1.When no benomyl was added to the medium, the highest radial growth rate of 7.15 mm/day was recorded for P. palmivora and this was significantly different (P< 0.05) from values obtained when benomyl was incorporated into the medium.Growth rates of P. palmivora were significantly reduced with increasing concentrations of benomyl with the lowest value (1.60 mm/day) being recorded when the fungicide was used at 150 ppm.With C. gloeosporioides, the highest growth rate of 2.95 mm/day was obtained when the medium was devoid of benomyl.In general, increasing concentrations of benomyl significantly reduced the growth rate of the fungus and the lowest growth rate of 1.55 mm/day was recorded on medium with 150 ppm benomyl.This value was similar to the growth rate of P. palmivora at the same concentration of benomyl (Table 1).At 7 days, C. gloeosporioides was present at the centre and the middle but generally not at the peripheries of colonies growing on plates containing 0 to 125 ppm benomyl (Table 2).By 14 days, the fungus had advanced  2 Benomyl concentrations of the original GCMA plates from which subcultures were made.
to the periphery of colonies on the benomylfree plates but was absent from the periphery of colonies on plates containing 75 to 125 ppm benomyl.It was also present at the peripheries of colonies on plates containing 150 ppm benomyl and was constantly detected at that location as the colonies grew.In general, C. gloeosporioides was detected at the peripheries of all plates by 21 days (Table 2).On the other hand, P. palmivora was detected at the centre, middle and peripheries of colonies grown on all plates, irrespective of the concentration of benomyl used and colony age (Table 2).
When sub-culturing was done at day 7 from the peripheries of colonies on 0 to 125 ppm benomyl plates onto benomyl-free media, C. gloeosporioides could not be detected on any of the resulting cultures even after 3 weeks.It was, however, present on cultures originating from colony peripheries on plates containing 150 ppm benomyl (Table 3).

DISCUSSION
In the present study, it was thought that the principle of selective isolation (Tsao, 1987) could be used to clean up P. palmivora cultures contaminated by C. gloeosporioides.Selective isolation involves prevention or slowing the growth of the undesirable organism (in this case C. gloeosporioides) so as to promote growth of the desirable one (P.palmivora in the present study) and permit the desirable fungus to be isolated and cultured in a pure state.Because selective isolation is usually achieved by incorporating fungicides into the culture medium, it was assumed that use of the fungicide benomyl would completely suppress C. gloeosporioides on the culture medium, allowing P. pal-J.Microbiol.Antimicrob.mivora to grow and be isolated in a pure state.This was thought to be so because benomyl is known to be effective against Deuteromycetes (Agrios, 1997) to which C. gloeosporioides belongs (Alexopolous et al., 1996).Use of benomyl in the present study did not completely inhibit the C. gloeosporioides at the rate used (0 to 125 ppm).It rather slowed down the growth of the fungus relative to that of P. palmivora.The isolation of P. palmivora in a pure form from culture peripheries achieved in the current study, was therefore, not due to complete inhibition of C. gloeosporioides but rather due to the inherently faster growth rate of P. palmivora on the medium even though benomyl also slowed down P. palmivora.This enabled P. palmivora to grow out of the contaminated zone, permitting its isolation in a pure form when transfers were made from colony peripheries.Subculturing from the centre and middle of colonies did not result in pure cultures of P. palmivora, indicating that these areas were already contaminated by C. gloeosporioides.Thus, this procedure can be used to obtain pure cultures of P. palmivora contaminated by any fungus with a slower growth rate.
In this study, we have demonstrated that pure cultures of P. palmivora can be obtained even from culture plates devoid of benomyl.The basis of this is again the differential growth rate of the two fungi indicated above.However, on plates devoid of benomyl, sub-culturing from the colony peripheries ideally should be within 7 days when C. gloeosporioides was restricted to the centre and middle portions of colonies.To prolong the period of subculturing from colony peripheries, amending the medium with benomyl at a 75 to 125 ppm a.i. to further slow down C. gloeosporioides is recommended.At these benomyl concentrations, the growth of C. gloeosporioides is highly reduced, further delaying its growth into the P. palmivora portion of the colony.In all cases, however, the interval of sub-culturing from colony peripheries should not exceed 14 days.If plates are kept for 3 weeks before subculturing, the possibility that C. gloeosporioides would have completely contaminated plates would be high and obtaining pure cultures of P. palmivora, even from peripheries of such cultures, would be problematic.This explains why in the past it has been impossible to obtain pure cultures of P. palmivora from any portion of overaged cultures.
At 150 ppm benomyl concentration, we were unable to get a pure culture of P. palmivora even from the peripheries of mixed cultures because at this concentration, the growth rates of both P. palmivora and C. gloeosporioides were not significantly different (1.60 mm/day for P. palmivora and 1.55 mm/day for C. gloeosporioides).Thus, contrary to Ritchie (2002), benomyl cannot be used under all conditions to selectively isolate fungi in the phylum Oomycota.

Table 1 .
Radial growth rate of P. palmivora and C. gloeosporioides on GCMA plates at different concentrations of benomyl 1 .

Table 2 .
Presence (+) or absence (-) of P. palmivora and C. gloeosporioides at different locations on colonies grown on media with different concentrations of benomyl 1 .

or absence (-) of Phytophthora and Colletotrichum
1The colonies were established from a P. palmivora culture contaminated by C. gloeosporioides.2Cen = centre, Mid = middle, Peri = periphery of colonies.

Table 3 .
Detection of P. palmivora and C. gloeosporioides on cultures established from peripheral transfers from mixed cultures of P. palmivora and C. gloeosporioides 1 .