This is the first comprehensive survey of the species diversity of Trichoderma for a region within the temperate Southern Hemisphere. New Zealand makes an ideal target for such a survey because of the extensive historical collections of this genus from both native and human-modified ecosystems. From the 320 Trichoderma strains sequenced for the translation elongation factor-1α (tef1α) gene, in addition to the names associated with voucher specimens at the New Zealand Fungarium (PDD, Landcare Research), we recognise 71 Trichoderma species as present in New Zealand. Thirty-two species are reported for the first time from New Zealand and 14 of these appear to represent undescribed taxa. The New Zealand species are positioned across most Trichoderma clades, with terminal lineages related to T. viride, T. koningii and T. harzianum well represented. Of the 14 undescribed species, Trichoderma sp. “atroviride B”, a sister species to T. atroviride s.s., was the most commonly recovered species. Records of several species known only from fungarium specimens could not be confirmed by DNA analysis. Populations of Trichoderma in New Zealand are likely to represent a mixture of ancient indigenous lineages, more recent natural introductions, and species introduced as a result of human-mediated dispersal. Twenty-three Trichoderma species have been reported only from New Zealand or other Southern Hemisphere locations. The diversity of Trichoderma species in New Zealand, their phylogenetic relationships, distribution, ecology, and possible origins are discussed in this paper.

The colonisation of plant roots by biological control agents is dependent on abiotic factors one of the most important being soil pH The rhizosphere and endophytic colonisation of ryegrass and sweet corn roots by the biological control agent Trichoderma atroviride LU132 were assessed in a pot experiment with nonsterile soil at three different pHs (55 65 and 75) T atroviride LU132 colonised the roots of both plants regardless of the soil pH with 113147 x 106 CFU/g of dry rhizosphere soil (DRS) for ryegrass and 136350x105 CFU/g DRS for sweet corn T atroviride LU132 was able to colonise both plants endophytically regardless of soil pH However the isolate was recovered from entire ryegrass roots but only from the upper parts of sweet corn roots These experiments demonstrated that T atroviride LU132 colonised the rhizosphere and roots within a soil pH range common to most NZ soils which is a desirable trait for biological control agents

Biological control agents (BCA) are beneficial organisms that are applied to protect plants from pests. Many fungi of the genus Trichoderma are successful BCAs but the underlying mechanisms are not yet fully understood. Trichoderma cf. atroviride strain LU132 is a remarkably effective BCA compared to T. cf. atroviride strain LU140 but these strains were found to be highly similar at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between separately isolated strains prompted us to undertake a genome comparison study in order to identify DNA polymorphisms. We further investigated if the polymorphisms had functional effects on the phenotypes. The two strains were clearly identified as individuals, exhibiting different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Genome sequencing identified only one non-synonymous single nucleotide polymorphism (SNP) between the strains. Based on this SNP, we successfully designed and validated an RFLP protocol that can be used to differentiate LU132 from LU140 and other Trichoderma strains. This SNP changed the amino acid sequence of SERF, encoded by the previously undescribed single copy gene “small EDRK-rich factor” (serf). A deletion of serf in the two strains did not lead to identical phenotypes, suggesting that, in addition to the single functional SNP between the nearly clonal Trichoderma cf. atroviride strains, other non-genomic factors contribute to their phenotypic variation. This finding is significant as it shows that genomics is an extremely useful but not exhaustive tool for the study of biocontrol complexity and for strain typing.

Soil-borne plant pathogens such as Rhizoctonia solani (Kuhn), Pythium ultimum (Trow) and Sclerotinia trifoliorum (Eriks) can reduce grass and forage legume establishment. The potential for biocontrol of these pathogens by Trichoderma fungi was evaluated. Following dual culture assays, nine Trichoderma isolates (five of Trichoderma atroviride and one each of Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma viride and Trichoderma virens) were chosen for assessment in pot experiments. In the presence of R. solani, perennial ryegrass (Lolium perenne L.) emergence was increased by 60–150% by two isolates of T. atroviride and by 35–212% by the isolate of T. virens, with the increase depending on growing medium and amount of pathogen inoculum. Red clover (Trifolium pratense L.) emergence in the presence of S. trifoliorum was significantly increased by two T. atroviride isolates and the T. hamatum isolate. In the presence of P. ultimum, white clover (Trifolium repens L.) emergence was increased by 25–42% by one isolate of T. atroviride and the T. hamatum isolate. However, for all three pasture species, some Trichoderma isolates reduced seedling emergence. Seedling growth (shoot and root fresh weight/plant) of the three pasture species was significantly increased by one or more T. atroviride isolates. On the basis of these results for both disease reduction and growth promotion, four T. atroviride isolates were selected for field assessment as biocontrol agents of soil-borne pathogens of pasture species.

Trichoderma spp. from New Zealand soils were evaluated (in greenhouse experiments and one field trial) for Rhizoctonia disease suppression and plant growth promotion of potato plants. Trichoderma virens LU549, T. atroviride LU144 and T. barbatum LU1482 demonstrated the greatest suppression of cankers on potato plants; the percentage of diseased stolons was reduced by 41–46%, compared with the R. solani control. Trichoderma virens LU549 also increased average tuber weight by 210%, and T. atroviride LU144 by 146%, compared with the R. solani control (in which tuber formation was highly suppressed). In plant growth promotion pot trials, the greatest proportional increases for three plant growth parameters (compared with the untreated control) were elicited by: T. harzianum LU1491 (number of tubers), T. barbatum LU1489 (total tuber weight), and Trichoderma sp. 792 LU1483 (average tuber weight). All six of these strains were selected and evaluated in all combinations in a 26 factorial greenhouse experiment. Trichoderma atroviride LU144 had positive impacts on several Rhizoctonia disease and plant growth parameters. Four of the strain combinations were subsequently tested in a field trial during the 2011/12 growing season, in which two Trichoderma strain combinations increased potato tuber yields. This research has shown potential for use of New Zealand Trichoderma strains to suppress Rhizoctonia diseases of potato and increase crop productivity.

Eleven plant species representing two native ecosystems (1) podocarp forest (Dacrycarpus dacrydioides, Plagianthus regius, Pittosporum eugenioides, Cordyline australis, Melicytus ramiflorus, Coprosma robusta and Asplenium gracillimum) and (2) grassland (Poa cita, Chionochloa rubra, Chionochloa rigida and Festuca novae-zelandiae) and representatives of a wider beneficial microbe population (arbuscular mycorrhizae (AM) and Pseudomonas spp.) were studied to assess possible non-target effects of a commercial T. atroviride product. Comparison of several physical markers (plant height, basal diameter, total leaf number, total leaf area, and fresh and dry weight of leaves, shoots and roots) showed that this T. atroviride isolate had no negative effect on plant health. Although photosynthetic pigment analysis indicated significant differences in chlorophyll and carotene levels between the Trichoderma and control treatments for some plants, this variation was not supported by physical changes in plant health. Culture-dependent and -independent analysis of AM fungi and Pseudomonads demonstrated that T. atroviride had no effect on these potential plant beneficial taxa in either ecosystem. The findings from this study suggest using Pittosporum eugenioides and Pagianthus regius for assessing the impact of imported microbial biocontrol agents on the plant growth of established podocarp forest plants, and Cordyline australis and P. regius for assessing the impact of these agents on seedling establishment. In tussock grassland ecosystems, Festuca novae-zelandiae and Poa cita are suggested for growth impact assessments, and P. cita and Chionochloa rubra for seedling establishment trials. The use of a combination of basic culture-dependent and -independent techniques for assessing changes in soil microbial communities often associated with plant health is also suggested.

Six field trials evaluated different methods of controlling onion white rot (OWR) on spring onions grown for 12-14 weeks in sandy soils naturally infested with sclerotia of Sclerotium cepivorum. The standard fungicide procymidone, applied using a variety of methods, did not provide satisfactory control of OWR at high disease sites. In contrast, the fungicides boscalid and tebuconazole provided very effective control of OWR. The fungicides azoxystrobin and triadimenol were also identified as having potential for OWR control on spring onions. Synthetic DADS (78% diallyl disulphide), injected into soil before growing spring onions, was very effective in reducing the population of sclerotia in soil and OWR incidence. Two applications of DADS at 5 or 10 L per ha were more effective than one application for reducing OWR incidence. Control of OWR was more effective when DADS was integrated with fungicide (boscalid). The biological control agent Trichoderma atroviride (C52) was more effective at reducing disease incidence under low than high disease pressure conditions. The information collected has enabled the development of an integrated strategy for the control of OWR on spring onions. The new control measures and integrated strategy are discussed.

Pellet and solid substrate formulations combined with a soil drench formulation of Trichoderma atroviride were evaluated under varying disease pressures in field trials to determine which formulation(s) provided the best control of the soil-borne disease onion white rot. Under low (11.8%) (average Area Under the Disease Progress Curve (AUDPC): 5.94%), moderate (37.7%) (average AUDPC: 11.26%) and high (64.4%) (average AUDPC: 24.76%) disease pressure, the standard fungicide programme (procymidone coated seed plus tebuconazole foliar spray) gave statistically significant disease control (76.3%, 91.3% and 58.8%, respectively) compared with the untreated control. Under low and high disease pressure, significant control, equal to the fungicide treatment, was achieved with the T. atroviride pellet plus soil drench treatment (72.0 and 39.6%, respectively). However, no Trichoderma formulation gave significant control of onion white rot under moderate disease pressure (20.7–34.2% control). When disease progress was examined over time for each trial, it was evident that the onset of disease occurred later in the growing season at the moderate disease pressure site (3 months after sowing) compared with Trial 3 at the high disease pressure site (2 months after sowing). At the low disease pressure site, the low level of inoculum and disease incidence enabled T. atroviride to bring about disease control. These results indicated that the performance of T. atroviride may be influenced as much by the timing of disease onset as the absolute disease pressure.

Trichoderma atroviride Karsten strain C52 populations were measured in agronomic soils in combination with soil organic amendments, a nitrogen fertiliser, fungicides and diallyl disulphide (DADS) (a Sclerotium cepivorum sclerotial germination stimulant). Trichoderma atroviride C52 populations did not proliferate 2 weeks after inoculation in sandy soils (4.7 x 10(2) cfu/g soil) compared with silt loam soils (1.1 x 10(6)-5.7 x 10(7) cfu/g soil), however, the addition of two blended pellet products containing poultry manure and other organic nutrients or humic acid plus organic matter to the sandy soil enabled populations to proliferate well (1.1-1.3 x 10(5) cfu/g soil). In vitro, twice field rate applications of urea reduced in vitro Trichoderma atroviride C52 growth (spore germination, germ tube length and mycelial growth), however, T. atroviride C52 populations were less sensitive to field rate applications of urea in field soil. Overall, T. atroviride C52 populations were not adversely affected when exposed to any of the fungicide soil treatments tested (populations ranged from 1.7 x 10(5)-1.1 x 10(8) cfu/g soil). Volatiles of DADS reduced T. atroviride C52 mycelial growth in vitro when DADS was applied to the medium at standard and twice recommended field rates but not at half field rate. No spore germination occurred under any of the DADS in vitro treatments compared with 100% spore germination in the control. However, when DADS was applied to soil 4, 6 and 8 weeks before application of T. atroviride C52, populations were unaffected. Based on these data, an application strategy for the use of a commercial formulation of T. atroviride C52 in an integrated white rot management programme for onions is proposed. (C) 2011 Elsevier Ltd. All rights reserved.

The onion industry in New Zealand incurs severe losses from white rot disease caused by the soil-borne pathogen Sclerotium cepivorum. This fungus produces hardy resting structures called sclerotia, which can remain in soil for many years. A fungal agent Trichoderma atroviride LU132 was shown to control infection by the pathogen and formulations of it provided a 65-70% disease control. This chapter provides a personal perspective on the factors that must be considered when making the decision of how much biocontrol is enough.

Sclerotium cepivorum sclerotia require incubation in soil to overcome constitutive dormancy a condition where the sclerotia will not germinate even when stimulated In Trial 1 artificial onion extract diallyl disulphide (DADS) was used to stimulate sclerotial germination of laboratory produced sclerotia after 1 2 and 3 month conditioning periods when incubated in two different soil types The results showed that soil type and fungal isolate did not affect dormancy and that approximately 16 33 and 21 of the sclerotia germinated after 1 2 and 3 month conditioning periods respectively In Trial 2 DADS significantly increased sclerotial germination compared with the control after 2 3 4 5 and 6 month conditioning periods Sclerotia required 6 months in soil before high rates of germination occurred (>89) when stimulated When a natural population of sclerotia (8 weeks old) (Trial 3) was exposed to DADS 51 of the population germinated compared with 21 in the control (Plt;0001)

The rhizosphere competence of the biological control agent Trichoderma atroviride isolate C52 was studied on onion roots both in the glasshouse and in the field when introduced into soil in a range of formulations. Proliferation of T. atroviride in the rhizosphere was formulation-dependent. A pellet formulation maintained the fungal concentration at 10(5) cfu per g soil, whereas solid-substrate and seed-coating formulations gave concentrations of 10(4) and 10(1) cfu per g soil, respectively. To facilitate rhizosphere-competence studies, a UP-PCR band profile generated with primer L45 for isolate C52 was used to enable conclusive identification of T. atroviride C52 when recovered from soil. When isolate C52 was introduced into Sclerotium cepivorum-infested soil as both pellet and solid-substrate formulations, there was no statistically significant difference in the disease control between these treatments, but the pellet treatment doubled the percentage of healthy plants compared with the control treatment.

A 10 year research programme at Lincoln University, investigating the use of Trichoderma species for biological control of soil-borne diseases of vegetable crops, has resulted in the development of two commercial products. TrichodryTM 6S and TrichoflowTM 6S based upon Trichoderma hamatum isolate 6SR4, are used to control Sclerotinia lettuce drop disease. The Trichodry 6S product is formulated as a dry flake, which is incorporated into nursery seedling mix and the Trichoflow 6S is a wettable powder which is used as a top-up drench before planting. The treatment stimulates seedling establishment and vigour and protects the developing seedling from Sclerotinia minor infection after transplanting in the field. The second commercial product is TrichopelTM Ali 52. Based upon Trichoderma atroviride isolate C52, which is used to control Sclerotium cepivorum, the causal agent of Allium white rot disease. The product is formulated as a granule and applied into the furrow at planting time. The fungus proliferates in the rhizosphere region and protects the growing seedling from pathogen attack by a combination of nutrient competition, antibiosis and mycoparasitism. The use of Trichopel Ali 52 under low to medium disease pressure in Pukekohe, the main vegetable growing region of New Zealand, gave a three fold cost benefit through yield increases in the 2003-2004 season. Current field development work involves the use of a wettable powder formulation of T. Atroviride distributed via a T-tape irrigation system to target mid-season applications of the product to the onion roots. Both products perform well under low to moderate disease pressure but, when there is high disease pressure, an integrated programme is required to give satisfactory control. Current research is focused on gaining a greater understanding of the biotic and abiotic factors, which influence biocontrol activity under field conditions as a means to enhance integrated control approaches. For example, T. Atroviride C52, when applied to the planting furrow, is co

The biocontrol isolate Trichoderma atroviride M1057 and a transformed hygromycin resistant biotype (M1057hygR) were compared using biological control rhizosphere competence and antibiosis studies to determine whether the transformed biotype performed in a similar manner to the wildtype strain In an onion growth chamber trial using soil naturally infested with the onion white rot pathogen Sclerotium cepivorum there was no significant difference (P>005) in the level of disease control given by the two T atroviride strains Similarly populations of T atroviride M1057 and M1057hygR were equivalent (P>005) in the rhizosphere of onion seedlings There was no significant difference (P>005) between the mycelial growth rates of S cepivorum when grown on agar amended with culture filtrate of T atroviride M1057 and M1057hygR Thus T atroviride M1057hygR has similar biological attributes to the wildtype isolate and can be used in future field studies looking at the population ecology of the biological control agent

The effect of Coniothyrium minitans on Sclerotinia sclerotiorum and Ciborinia camelliae sclerotial viability was determined on three different substrates: sand, soil and sawdust using fully factorial, repeat experiments (Trials 1 and 2). In Trial 1, C. minitans significantly reduced the number of viable S. sclerotiorum sclerotia in sand (48%) and sawdust (0%) but not in soil (60%) compared with the untreated sclerotia (92, 64 and 88%, respectively) after 8 weeks. Although C. minitans had no effect on C. camelliae sclerotial viability, the sawdust only treatment reduced viability to 0% after 4 weeks. In the repeat experiment (Trial 2), C. minitans had no effect on S. sclerotiorum or C. camelliae sclerotial viability, although C. camelliae sclerotial viability was again significantly reduced in the sawdust control treatment (8-12%) compared with the sand and soil control treatments (> 84%). Coniothyrium minitans has some potential for biocontrol of S. sclerotiorum, but not of C camelliae. Sawdust may be an option for use as an under plant mulch for control of C. camelliae.