CHAPTER 4: DISCUSSION
4.3 Optimising different methods of decontamination
4.3.5 Biocontrol agents and their effects
4.3.4.4 Effect of Trichoderma isolates as conidial suspensions on Trichilia
Although different strains and species of Trichoderma have been used as biocontrol agents for many crops against many plant contaminants, no other work has been done on their effect in vitro on axes excised from recalcitrant seeds. In view of the significant problems posed by fungi in embryo culture, the present investigation aimed at testing the effect of EcoT and Eco77 on germination, as well as on the contamination status of embryonic axes of T. dregeana in vitro.
a) Effect of
Trichoderma on the viability of the embryonic axes of Trichilia dregeanaplated on water agar
Inoculum of Trichoderma, introduced as a spore suspension in water, was co-cultivated with the embryonic axes on water agar for 24 to 72 h. After 24 h, no Trichoderma penetration of axes had occurred, and, even when the co-cultivation time was increased, only a few were revealed as having been penetrated by the biocontrol agent. This might have been the result of the high spore concentration – which is in agreement with the inhibition of germination of Trichoderma hamatum spores on water agar, especially when the spore count was more than 108 spores mL-1 (Nol and Henis, 1987). Although in the present study 1x106 conidia mL-1 were introduced, a contributory factor might have been that germination of Trichoderma spores, even in lower concentrations than 1x108 mL-1, is retarded on water agar due to lack of nutrients. The minimum period taken for the conidia of the Trichoderma spp. to germinate is >10-14 h at 26°C on a nutrient rich medium (Lifshitz and Baker, 1986). However, there was a negative effect on the vigour of embryonic axes plated for longer periods with Trichoderma suspension. It is possible that competition between the axes and spores for space on the agar surface might have affected the vigour of the co-cultivated embryonic axes. It is also possible – that some material exuded from the ungerminated spores could have had adverse effects on the co-cultured axes. However, both these explanations are necessarily conjectural at this stage.
Axes were plated on water agar which had been previously inoculated with Trichoderma for 7 d, for 24 to 72 h. All axes showed Trichoderma penetration after 24 h, but axis vigour and viability were seriously compromised. The negative influence on the growth and development of the axes
might have been as a result of the organic acids secreted by the Trichoderma strains, viz.
gluconic, citric or fumaric acid which acidify the surrounding environment (Gómez-Alarcón and de la Torre, 1994). The present results are in agreement with the studies on potted maize (Zea mays L. Cultivar B73) in which no seedlings emerged from the soil which was previously inoculated with various isolates of Trichoderma (T. harzianum [T969], T. harzianum [T447], T.
hamatum [T614], T. roseum [T678], Gliocladium virens [G525] and an unknown Trichoderma species isolate [Trichoderma sp. T]) (Hajieghrari, 2010). As seedlings from the seeds potted in non-inoculated soil emerged after 30 d, that author suggested that the bio-control agents could actually have become parasitic (see below) after seed germination in the inoculated soil, resulting in pre-emergence damping-off (Hajieghrari, 2010).
b) Embryonic axes immersed in Trichoderma spore suspension and then cultured on half strength MS medium
Embryonic axes were immersed in Trichoderma conidial suspensions for 30 min and then co- cultivated (i.e. axes together with adherent spores) on half stength MS medium for times from 24 to 72 h. Axes germinated readily on the half strength MS medium after immersion in the spore suspension for 24 h, but after immersion for 48 h and 72 h the germinability of the axes was compromised. However, although after the 24 h immersion period all axes appeared to have been inoculated with Trichoderma, it was also noted that there was reduction, but not complete elimination of the inherent contamination compared with the control axes (Table 3.35). This result is in agreement with the trials conducted on the pruning wounds of vine against Eutypa lata by Eco77, where it was recorded that after 24 h, colonisation of the vine by the just-germinated Trichoderma spores was minimal with consequent protection against the contaminant being inadequate10.
Some studies on the effects of various species of Trichoderma have indicated that they have the potential to increase seed germination totality (germination percentage) and productivity of the resultant plants, including maize (Harman, 2006) pigeon pea (Manju and Mall, 2008); cotton seedlings (Hanson, 2000); rice (Mishra and Sinha, 2000); Okra (Mukhtar, 2008); Cabbage and Lettuce (Rabeendran et al., 2000); chickpea (Dubey et al., 2007). Several other studies also reported that T. harzianum, T. viride and T. pseudokoningii not only promoted seed germination, but also improved the vigour of the pea (Zheng and Shetty, 2000), and watermelon (Bharath et al., 2006), seedlings with diminished occurrence of seed-borne fungal contaminants. In all these cases a symbiotic relationship between the Trichoderma species and the treated seeds/seedlings is
assumed. However, there are also examples of Trichoderma spp. becoming parasitic rather than a symbiotic (Hajieghrari, 2010) when the conidial suspensions (106-107 spores per mL-1) of Trichoderma harzianum T969, T. harzianum T447, Trichoderma hamatum T614, Trichoderma roseum T678, Gliocladium virens G525 and the unknown Trichoderma species isolate (Trichoderma sp. T) were used to treat the seeds. In that study, it was found that seed germination as well as the rootlet and shoot development of maize caryopses (Zea mays L. cultivar B73) was adversely affected, apparently by necrosis of the inner seed tissues by Trichoderma (Hajieghrari, 2010). That author reported that culture filtrates of the various species of Trichoderma also had adverse effects (see above).
The inhibitory effect of Trichoderma has been further documented in various other studies, e.g.
the effects of Trichoderma spp. on maize seed and seedlings (Sutton, 1972; Mc-Fadden and Sutton, 1975) and also on cucumber, pepper and tomato seedlings (Menzies, 1993). Most of the Trichoderma spp. penetrate only on the root epidermis without causing damage and the further penetration of the hyphae is suggested to be blocked by the deposition of callose by the neighbouring cells (Yedidia et al., 1999). But in the present study even at 24 h of co-cultivation with the Trichoderma, retarded growth and reduced biomass of the T. dregeana axes was recorded. This could be due to the penetration of the hyphae of Trichoderma into the tissues below the root epidermis of the germinating axes, on the assumpton that blocking by callose was inadequate. Alternatively, inhibition of axis germination could have been due to increasingly anoxic conditions developing during axis immersion, despite the process having been carried out on a shaker. It must be stressed that axes excised from recalcitrant seeds are metabolically-active (Berjak and Pammenter, 2004; 2008), hence they would be sensitive to anoxia, in contrast to orthodox seeds, which would be metabolically inert at least early in an immersion period.
In the current study it was shown that if the Trichoderma was not killed (by application of Benlate) once successful inoculation was achieved, the fungus would overgrow and kill the embryonic axes.