Towards understanding the ecology and mechanisms of biocontrol of Clonostachys rosea IK726

Clonostachys rosea (syn. Gliocladium roseum) IK726 was originally selected as an effective biocontrol agent (BCA) against cereal seed borne diseases caused by Fusarium culmorum and Bipolaris sorokiniana. We have studied the efficacy of the antagonist against different pathogens in several crops and found that the antagonist also is able to control Alternaria radicina and A. dauci on carrot seeds and different cold-storage fungi in acorns. IK726 is also able to reduce severity of soil borne Pythium spp. in cabbage, carrot and sugar beet. In addition, growth-promoting effects of IK726 have been demonstrated in barley and tomato. In order to develop and improve application methods and control strategies, essential basic studies of ecology and the mechanisms of control of IK726 is needed and has led us to use various molecular tools. The UP-PCR technology is used for strain recognition and we have developed GUS and GFP-transformants that resembles the wildtype strain in ecological fitness parameters. Using either the GUS-transformant or UP-PCR we have found that IK726, when applied with seeds, reproduces and survives several months in the rhizosphere of field grown barley and carrot. The GFP-transformant is used to study the behavior and in situ interactions of the antagonist with pathogens and plants. Using the GFP marker, we have observed conidial germination, colonization and conidiogenesis in natural soil, in vermiculite and on carrot and barley seed and roots and on barley leaves. Moreover in situ interactions with Alternaria on carrot material have been studied. The modes of action of C. rosea are not well understood but enzymatic activity, mycoparasitism, substrate competition, antibiosis and induced resistance are thought to play a role. Barley treated with C. rosea IK726 has an enhanced chitinolytic and glucanolytic activity compared to the activity in non-treated barley in pot experiments with field soil. Identification of chitinases from IK726 and studies of their role in interactions with pathogens have therefore been addressed in a recently initiated project. Preliminary results indicate that IK726 produces three types of chitinases, which seem to be regulated by glucose. Development of degenerated primers for cloning of an endochitinase is in progress.

Universally Primed PCR (UP-PCR) and its applications for taxonomy in Trichoderma

Universally Primed PCR (UP-PCR) is a PCR fingerprinting method that has demonstrated its applicability in different aspects of mycology. These applications constitute analysis of genome structures, identification of species, analysis of population and species diversity, revealing of genetic relatedness at infra-and inter-species level, and identification of UP-PCR markers at different taxonomic levels (strain, group and/or species) . A further development of the UP-PCR technique is an UP-PCR product cross hybridisation assay that facilitates investigation of sequence similarity (homology) of UP-PCR products and grouping of strains into UP-PCR hybridisation groups. This separates the strains into entities with high genetic similarity (DNA homology) . UP-PCR has been used as an aid in taxonomy and species delineation, and to monitor biocontrol strains following their release into the environment by fingerprint characterisation of pure cultures and through direct detection in soil by amplification of UP-PCR-derived SCAR markers. The technique has been applied to Trichoderma strains in particularly with the aims of strain recognition and classification.

Novel understanding of Trichoderma interaction mechanisms

Trichoderma- based biofungicides are a reality in commercial agriculture, with more than 50 formulations registered worldwide as biopesticides or biofertilizers. Several research strategies have been applied to identify the main genes and compounds involved in the complex, three-way interactions between fungal antagonists, plants and microbial pathogens. Proteome and genome analyses have greatly enhanced our ability to conduct targeted and genome-based functional studies. We have obtained reproducible 2-D maps of the entire fungal proteome in various conditions of interaction, which permitted the isolation of many proteins related to specific functions. Many differential proteins from several biocontrol strains of Trichoderma spp. during the in vivo interaction with different plants and/or several phytopathogenic fungi have been isolated and analyzed by MALDI-TOF. Relevant genes have been cloned and specifically inactivated, to demonstrate their function in biocontrol and induction of disease resistance. GFP-based reporter systems with interaction-inducible promoters allowed the characterization of regulatory sequences activated by the presence of the pathogen or the plant. From extensive cDNA. and EST libraries of genes expressed during Trichoderma-pathogen-plant interactions, we are identified and determined the role of a variety of novel genes and gene-products, including ABC transporters specifically induced during antagonism with other microbes; enzymes and other proteins that produce or act as novel elicitors of Induced Resistance in plant and promote root growth and crop yield; proteins possibly responsible of a gene-for-gene avirulent interaction between Trichoderma and plants; mycoparasitism-related inducers released from fungal pathogens and that activate biocontrol in Trichoderma; fungal promoters specifically induced during mycoparasitism and plant colonization; plant proteins and a novel phytoalexin induced by the presence of the fungal antagonist; etc. We have also transgenically demonstrated the ability of Trichoderma to transfer heterologous proteins into plants during root colonization. Finally we have used GFP and other markers to monitor the interaction in vivo and in situ between Trichoderma and its host (s) (the fungal pathogen and the plant).

Establishment and Growth of Potato Micro-Cuttings in Sand Trays

Two separate experiments were conducted to evaluate the success of the establishment and growth of micro-cuttings of potato (5 - 6 cm tall) in sand trays [38 cm (L) × 28 cm (W) × 7.5 cm (H) plastic trays] under controlled environment (22?C ± 2?C, 60 - 75 μmosm–1?s–1 light energy for 16 h daily). In the first experiment, micro-cuttings of potato cv. Diamant were planted at six populations (500, 600, 700, 800, 900 and 1000 cuttings per tray) in treated sand (sun dry, 1% formaldehyde, 0.2% Dithane M-45 and control). The mortality percentage of micro-cuttings was nil for sun dry sand while formaldehyde and dithane M-45 treated sand had 1% - 4% against 15% in the control with the highest population density. Mortality of micro-cuttings in formaldehyde and dithane M-45 treated sand trays were found not to be related to pathogenic organism rather toxic effect of these two chemicals. Micro-cuttings in Sun dry and control treatments showed better growth performance than these in chemically treated sand trays. In the second experiment, urea @ 1, 2 and 3 g per tray was applied as solid form after 15 days of planting the micro-cuttings and as liquid form @ 0.5, 1 and 2% solution sprayed in the micro-cuttings repeatedly after 15, 30, 45 and 60 days of planting. The micro-cuttings which received urea as solid state died within 2 - 3 days and 2% urea solution was also detrimental. Urea solution @ 0.5% found to be very effective for vegetative growth of micro-cuttings in sand trays. The control was also good for vegetative growth but at a slower rate.

The Influence of Organic Manure and Biochar on Cashew Seedling Performance, Soil Properties and Status

A nursery experiment was conducted at Cocoa Research Institute of Nigeria to evaluate the impact of manure on cashew seedlings. Treatments consisted of biochar, compost, combination of biochar + compost and a control. Treatments were applied at rate of 0 g (control), 5 g compost, 5 g biochar and 2.5 g each of biochar and compost combination into 5 kg soil in pots, laid out in a completely randomized design (CRD) with three replications. The experiment was monitored for four months in the greenhouse. Data were collected on number of leaves, plant height, stem girth, soil nutrient composition and nutrient uptake, all analyzed using analysis of variance. The treated soils were also assayed on Potato Dextrose Agar for associated mycoflora. The study showed that the soil PH, N, P, K, Ca and Mg were equally enhanced significantly relative to the control, while Aspergillus, Trichoderma, Beauverie, Penicillium, Lasiodiplodia and Rhizopus cultured treated soils shows that Trichoderma harzianum and Beauverie bassiana were significantly higher in combination of biochar and compost treated soil and posed that their bio-control potential is coupled with soil fertility enhancement characteristics. The results also indicate that stem girth and number of leaves were significantly (P ≤ 0.05) different with respect to applied treatment at the first month after treatments application. The leaf area was not enhances with the application of biochar. Combination of biochar + compost significantly enhanced number of leaves and stem girth at the 4th and 12th weeks after planting. Similarly leaf K and P uptake were significantly (<0.05) increased by the application of organic compost and biochar irrespective of whether combined or not compared to the control seedlings. The effect of biochar and compost on cashew leaf Ca and Mg uptake were not significantly different from the control but had a higher value relative to the control. It could therefore be recommended that addition of compost and biochar for sustainable production of cashew seedlings in the nursery be embraced by prospective cashew farmers and seedlings distribution in Nigeria.