agents
A. Stewart{ XE "Stewart, A." }, Y.W. Workneh and K.L. McLean
Bio‐Protection Research Centre, PO Box 84, Lincoln University, Canterbury 7647, NZ
INTRODUCTION
The influence of environmental factors such as pH, moisture,
temperature and other abiotic factors such as fertiliser and
pesticide application on the establishment, proliferation and
persistence of biocontrol agents in the field has been intensively
investigated (1). However, there is little understanding of the
nature of biotic influences which are likely to play an equally
important role in determining the nature of the biocontrol
outcome. This paper reports on a preliminary study that
examines the effect of common soil microbes on two biocontrol
agents, Trichoderma atroviride LU132 active against onion white
rot and Trichoderma hamatum LU593 active against Sclerotinia
lettuce rot (2).
MATERIALS AND METHODS
Soil microbes. Forty‐eight microbes representing 11 fungal
genera (Acremonium, Alternaria, Aspergillus, Beauveria,
Chaetomium, Cladosporium, Fusarium, Metarhizium,
Paecilomyces, Penicilllium, Verticillium), seven bacterial genera
(Agrobacterium, Azotobacter, Bacillus, Burkholderia,
Flavobacterium Paenibacillus) and four actinomycete genera
(Actinomyces, Arthrobacter, Rhodococcus, Streptomyces) were
obtained from NZ culture collections (Lincoln University,
Landcare Research, AgResearch).
Dual culture assays. Test microbes were inoculated 3d prior to
or simultaneously with the Trichoderma on 9 or 15cm diameter
PDA plates. An inoculum plug of the test microbe was placed
3cm apart from the Trichoderma in the centre of the plate.
Colony interactions were monitored every 24h until Trichoderma
colony growth stopped or was constrained by the edge of the
plate. Trichoderma colony area (mm2) was measured and
percentage inhibition compared to the Trichoderma control
calculated.
Soil pot assays. Inocula of six test microbes were produced on
rice grains and incorporated into Templeton silt loam soil in pots
to give 106 cfu/g soil. Trichoderma was applied to the soil (106
cfu/g soil) as a granular formulation (Agrimm Technologies Ltd).
Pots were incubated at constant temperature and moisture for
30d. At weekly intervals, soil samples were taken from three
random spots in each pot and Trichoderma population counts
(cfu/g soil) determined using soil dilution plating on Trichoderma
selective medium.
Statistical analyses. Data was analysed using one‐way ANOVA
and treatment means compared using Fishers LSD.
RESULTS
Dual culture assays. Co‐culture on PDA revealed six fungi and
one bacterium that significantly inhibited Trichoderma colony
growth (Table 1). Greatest inhibition (>85%) occurred with Aspergillus niger and Paecilomyces lilacinus for T. atroviride and
T. hamatum, respectively. In general, T. hamatum was less
sensitive than T. atroviride to the test microbes, in particular to C. globosum.
Soil pot assays. T. atroviride populations were significantly
reduced in soil treated with Alternaria, Aspergillus, Metarhizium,
Paecilomyces and Daldinia (Fig. 1) T. hamatum was less sensitive
to the test microbes but the trend was similar (data not shown).
Table 1. Percentage inhibition of Trichoderma colony growth after 10d
dual culture with test microbes
Test microbes T. atroviride T. hamatum
Alt. alternata 94.7 a* 72.8 c P. lilacinus 84.6 b 85.1 a Asp. niger 96.5 a 82.2 ab C. globosum 87.2 b 44.5 d M. anisopliae 82.5 b 74.2 bc D. eschscholzii 56.4 c 51.2 d Agrobacterium sp 67.1 c 31.3 e * Values within columns followed by the same letter are not significantly different. 0.00E+00 2.00E+04 4.00E+04 6.00E+04 8.00E+04 1.00E+05 1.20E+05 1.40E+05 A. a ltern ata A. n iger C. g lobos um M. a niso pliae P. lil acin us D. e schs chol zii Agro bacte rium Cont rol CF U /g ram so il
Figure 1. T. atroviride population (cfu/g soil) after 30d in soil treated with
different test microbes.
DISCUSSION
Six fungi and one bacterium significantly inhibited Trichoderma
colony growth on agar plates with differential sensitivity
observed between the two Trichoderma strains. Preliminary
studies suggest the inhibition is due to the production of
antifungal metabolites by the test microbes. The high inhibition
observed in culture was not reproduced in the soil assay where
five of the seven test microbes reduced Trichoderma populations
but only by ten‐fold. Since test microbe populations in the field
are likely to be lower than those used here, the results likely
overestimate the potential negative impact on Trichoderma
biocontrol agents applied to soil. However, further work
examining the effect of the test microbes in different soil types is
needed since metabolite production by the test microbes may
be influenced by soil abiotic factors.
ACKNOWLEDGEMENTS
Funding for this project was provided by the NZ Tertiary
Education Commission.
REFERENCES
1. Kredics L, Antal Z, Manczinger L, Szekeres A, Kevei F, Nagy E (2003) Influence of environmental parameters on Trichoderma strains with biocontrol potential. Food Tech. and Biotechnol. 41, 37–42. 2. Stewart A, McLean K L (2004) Optimising Trichoderma bio‐
inoculants for integrated control of soilborne disease. Proc 3rd
Australasian Soilborne Diseases Symposium 55–56.
Session
6C—Alternatives
to
chemical
control
Understanding Trichoderma bio‐inoculants in the root system of Pinus radiata
P. Hohmann{ XE "Hohmann, P." }A, E.E. JonesB, R. HillA, A. StewartA
A
Bio‐Protection Research Centre, PO Box 84, Lincoln University, Lincoln 7647, New Zealand
B
Department of Ecology, Faculty of Agriculture and Life Sciences, PO Box 84, Lincoln University, Lincoln 7647, New Zealand
INTRODUCTION
The genus Trichoderma are beneficial soil‐borne fungi and a well
known source of biological control agent active against a wide
range of crop diseases, including those of pine trees (1). Several
isolates of Trichoderma have been shown to improve
establishment and reduce pathogen infection of Pinus radiata in
the nursery and in forestry plantations (2). Three isolates of
different Trichoderma species were selected for this study. T. hamatum (LU592) and T. harzianum (LU686), known to stimulate
growth and improve establishment of P. radiata seedlings, and T. atroviride (LU132) which had no stimulatory activity. To enable
more predictable and effective use of Trichoderma bio‐
inoculants, their establishment and population dynamics was
determined. In addition, the effect of each isolate on P. radiata
seedling vitality and growth was assessed.
MATERIALS AND METHODS
Each Trichoderma isolate was applied either as a seed coat
formulation (4 x 105 spores/seed; SC) or a spore‐suspension (5 x
106 spores/pot; SA) sprayed directly after sowing the P. radiata
seeds. P. radiata seeds were grown in root‐pruning containers
for 7 months under conditions reflecting those used in the
commercial PF Olsen nursery. Health and growth assessments
included mortality rate, shoot height and shoot and root dry
weight measurements. During the 7 month trial period,
Trichoderma populations were enumerated in the bulk potting
mix, rhizosphere, rhizoplane and endorhizosphere subsystems
by dilution plating. At the 20 week assessment, recovered
Trichoderma colonies were identified using morphological and
molecular techniques to differentiate between introduced and
indigenous species. A large‐scale experiment was set up at the
PF Olsen nursery under commercial conditions to verify the
results for LU592.
RESULTS
T. hamatum LU592 performed the best out of the three
introduced isolates. Seedling mortality rate was reduced from
5.2% for the control to 0.2% for LU592 and 0.4% for T. harzianum LU686 SC. LU592 and LU686 SC also increased shoot
height by 17% and 11%, respectively. Results also indicated that T. atroviride LU132 increased the root/shoot ratio.
Trichoderma populations of all SA treatments were significantly
higher in the rhizosphere (by 2.1 to 3.3 times) compared with
the control. Applied Trichoderma spp. could be differentiated
from indigenous isolates by colony morphology and confirmed
by molecular sequencing. Introduced Trichoderma isolates could
be detected even though overall Trichoderma populations did
not reveal significant differences to the control. In the
rhizosphere, introduced isolates established with levels of ~20%
for LU132 SA and LU592 SC. T. harzianum LU686 was not
recovered from the rhizosphere after 20 weeks. T. hamatum
LU592, when spray applied, was the only isolate clearly
dominating all four subsystems bulk potting mix, rhizosphere,
rhizoplane and endorhizosphere.
T. hamatum LU592 as a seed coat and spray application
significantly increased shoot height, shoot and root dry weight
and stem diameter compared with the control in the large‐scale
experiment (Table 1).
Table 1. Increase (%) in P. radiata seedling growth parameters by T.
hamatum LU592 compared with the control. All values significant at P > 0.05 from the control.
Growth factor Seed coat Spray Suspension
Shoot height 7.4 9.5
Dry weights ‐ roots 17 21
‐ shoots 23 24
Stem diameter 9.0 9.4
number root tips 11 n.s.
n.s. = not significant
DISCUSSION
Both T. hamatum LU592 and T. harzianum LU686 increased the
growth of P. radiata seedlings, with the subsequent large‐scale
experiment confirming the growth promotion effects of LU592.
The spray application performed slightly better than the seed
coat application. This reduction in seedling morality and increase
in seedling growth represents a substantial economic benefit to
the industry.
The spray application method clearly promoted the
establishment of the introduced isolates in the root system of P.
radiata. T. harzianum LU686 was found to be an early
rhizosphere coloniser (declining after 12 weeks). Strong
rhizosphere competence was identified for T. hamatum LU592.
The ability of Trichoderma, LU592 in particular, to establish in
the rhizosphere and penetrate the roots is a crucial indicator of
beneficial activity (1).
LU592, being the most effective isolate at colonising all P.
radiata root subsystems, was selected for more detailed
ecological studies using a genetically marked strain. Future
experiments will focus on the use of a fluorescently marked
isolate of LU592 to verify rhizosphere competence, examine
spatio‐temporal distribution within the rhizosphere and
determine endophytic activity including interactions with
ectomycorrhizae.
ACKNOWLEDGEMENTS
This study is part of the Ecosystem Bioprotection program
LINX0304 funded by the NZ Foundation for Research Science and
Technology (FRST). We would like to thank PF Olsen for nursery
access.
REFERENCES
1. Harman, G.E., Howell, C.R., Viterbo, A., Chet, I. & Lorito, M. (2004). Trichoderma species—Opportunistic, avirulent plant symbionts. Nature Reviews Microbiology. 2(1): 43–56.
2. Hood, I.A., Hill, R.A., Horner, I.J. (2006). Armillaria Root Disease in New Zealand Forests. A Review. Review document written for the Forest Biosecurity Research Council.