Viticulture PENETRATION OF TRICHODERMA HARZIANUM INTO GRAPEVINE WOOD FROM TREATED PRUNING WOUNDS 1PLANTwise Services Ltd, PO Box 181 Lincoln, New Zealand2Agrimm Technologies Ltd, PO Box 35 Lincoln, New ZealandCorresponding author: [email protected]ABSTRACT
VinevaxTM (containing a mixture of strains of the fungus Trichoderma harzianum) is normally applied as soon as possible after grapevine
pruning. However, using a range of six timings after pruning from
immediate to 4 days later, it was shown that the best penetration of
the fungi into the wood was with treatment at approximately 5 h after
pruning. A novel method of measuring penetration and vigour of the
growth of Trichoderma spp. into grape wood is described. A second
trial using a range of concentrations of VinevaxTM (at and below label
rates) was applied as a general cover spray over dormant, pruned vines.
The percentage incidence and vigour of penetration of the T. harzianum
into canes was concentration dependent and lower than the targeted
hand-applied treatment earlier in the season. Therefore targeted, timely
applications of the product appear to be best for good wood penetration
Keywords: Trichoderma, grapevines, pruning, wood-invading fungal INTRODUCTION
The treatment of pruning and trunk reconstruction wounds of grapevines has become
important with the finding that these are often the portals for entry of many debilitating
trunk diseases (Adalat et al. 2000; Larignon & Dubos 2000). The main pathogens
involved are Eutypa lata, Botryosphaeria spp., Phomopsis spp. and the Phaeomoniella/Phaeoacremonium/Acremonium/Phialophora complex (Hunt et al. 2001; Halleen et al.
2005). Hunt et al. (2001) also showed that Trichoderma-based treatments gave an 85%
decrease in isolation of many of these fungi 8 months after pruning, while John et al.
(2004) in an in vitro study showed that Trichoderma-treated grape wood gave a 90%
decrease in colonisation by E. lata. Di Marco et al. (2004) showed that when Trichoderma
was applied immediately after pruning in late winter, around 90% of wounds yielded the
applied fungus 5 days after application and from 60–70% after 60 days.
Present label instructions for the Trichoderma harzianum-containing VinevaxTM
recommends application of the product immediately after making pruning cuts, but
the practicality of this timing in large vineyards and when using mechanical pruning
techniques becomes problematic. The purpose of the present study was to determine
the optimal time after pruning that a Trichoderma product should be applied and at
what dilution to still obtain a substantial colonisation of the wood using spur and cane
In Trial 1, VinevaxTM (Trichodermaharzianum formulated for pruning wound
application in vineyards) was applied at label rates (10 g/litre) at the following intervals
after pruning: 15, 30 and 60 min; end of day (3-4 h); next day (24 h); and 4 days later
New Zealand Plant Protection 59:343-347 (2006) 2006 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.htmlViticulture
(96 h). Untreated controls were sprayed with water 15 min after pruning. There was also
an additional treatment of the fungicide propiconazole (0.625 ml active ingredient/litre)
applied 15 min after pruning. Treatments began on 30 June 2005 with all treatments on
the day of pruning being applied with a hand-held trigger spray bottle while treatments
on the following day and 4 days later were applied with a knapsack sprayer. Each
treatment consisted of two vines spur pruned to 4 buds and two vines cane pruned. The
treatments were randomised through a 15-year-old block of Chardonnay at Lincoln
Thirty-three days after pruning, 8 spurs and 4 cane samples were harvested per
treatment by trimming approximately 10 cm off the spur (leaving a 2 bud spur) and cane
from the pruned and treated portions of the vines. The new pruning cuts were re-treated
with VinevaxTM following collection of the samples. The cuttings were then cool-stored
for 7 days until processing. From each vine four straight spurs and two cane-ends were
selected and cut to fit inside a 9 cm plastic Petri dish. The distal (inoculated) end of the
cane or spur was marked on the wood. A strip of bark was removed from two sides of
each sample and then sprayed with alcohol and flamed in a laminar flow cabinet. Using
flamed secateurs, each cane or spur was split longitudinally along the peeled surfaces by
cutting into the proximal (non-treated) end and pulling the two sides of the cane apart.
One half of each cane/spur was placed flat, cut side down onto the surface of a malt
extract agar (MEA) plate. The cut surface was laid as flat as possible on the agar surface
so that the entire cut area had good contact with the agar medium. Each plate was then
marked to indicate the spray inoculated (or distal) end of the sample. The plates were
incubated for 3 days at 25°C in the dark. The patterns of mycelium growing out from
the portions of split cane into the agar were marked with felt pen on the base of the
agar plates. The plates were then incubated for a further 3 days under near ultraviolet to
stimulate sporulation of the colonies of Trichoderma that grew out from the tissue. Plates
that demonstrated typical Trichoderma sporulation were then set aside and the original
markings denoting the 3-day old growth were measured for distance down the cane where
the colony originated and the radius of growth out from the source (Fig. 1).
In Trial 2, VinevaxTM was applied at the following three application rates to cane-pruned
vines in late August: 10 g/litre (label rate), 2 g/litre and 1 g/litre. Each treatment was
applied to one 15 m row containing 14-16 vines. Each vine had two canes and thus two
treated wounds for evaluation. The trial was located in non-adjacent Chardonnay rows
at the same location as Trial 1. Vines were pruned at 10 am and VinevaxTM at the above
rates applied at end of day (6 pm), 8 hours after pruning. The treatments were applied
using a Solo 475 knapsack with an E04-80 nozzle. This gave a consistent spray pressure
of 40 psi. The application rate was 1300 ml/min at a walking speed of 1.8 km/h. This rate
was required in order to apply sufficient spray to the vines and adequate wetting of the
wounds. From the above parameters, the spray rate was calculated at 173 litres/ha.
After 4 weeks, a 10 cm portion was cut from the treated end of each cane on the vines
in each row. Again, the pruning wounds were re-treated. These portions were assessed
for colonisation by the Trichoderma sp. as described in Trial 1.
Data obtained from the trials were subjected to ANOVA where appropriate.
The results from Trial 1 (Table 1) showed that pruned vines treated with VinevaxTM
either 4 h (end of the day) or 24 h (next day) after pruning gave the greatest incidence
of inoculation, distance down the cane and vigour of growth of the Trichoderma sp.
compared to those treated within 30 min or after 4 days.
The poor results from vines treated either 15 or 30 min after pruning may have been
due to vascular “bleeding” or leakage following cutting. This flow may have impeded
the growth or penetration of spores of Trichoderma into the wood. Once this had begun
to diminish, then penetration may have been more readily achieved by the applied
2006 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.htmlViticulture FIGURE 1: Typical pattern of growth of Trichoderma harzianum from a split spur portion from a VinevaxTM-treated vine after 3 days incubation in the dark and 3 days under near UV light. D = approximate distance down the cane that was the focus of growth out into the agar of the T. harzianum.R = the radius of growth of T. harzianum out from the cane at 3 days. TABLE 1: Trichoderma incidence (%), penetration (distance down cane from the pruning wound, mm) and vigour (growth radius out from cane into culture, mm) after treatment of pruned spurs and canes of grapevines with VinevaxTM at varying times after pruning in June.
The low scores when the VinevaxTM was applied after 4 days may have been due to
initiation of the healing process of the cut-surfaces at this time. This appeared to have
been greatest in the spur-pruned treatments compared to the cane-pruned. The difference
may have been due to the orientation of the cut surfaces. The horizontal cut surfaces of
the spur-pruned vines may have dried and “cured” faster than the vertically orientated
2006 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.htmlViticulture
It was noted that the Trichoderma-yieldingcane/spur portions in culture grew almost
no saprophytes or wood-invading pathogens. It is unclear if this was a reflection of
competition in the cane by the applied Trichoderma or a case of competition in the
culture plates. This observation may require further investigation.
The Trichoderma isolates yielded by the untreated controls (25% from canes and
12.5% from spurs) is probably a reflection of the background levels of this fungus in
the environment. They were also located less deep in the wood, probably reflecting less
Note that the above results are only applicable to spurs and cane-end pruning
wounds. The treatment may perform somewhat differently on large wounds created
when re-working cane architecture or top-grafting. This requires further investigation,
necessitating partial, or complete destruction of vines in the trial.
In Trial 2, Trichoderma spp. were isolated from only 35% of the cane portions at
the lower rates of application (one-fifth and one-tenth of label rate) and only 15% at
the highest rate (label rate) of application (Table 2). The incidence of recovery of the
Trichoderma sp. in August was low at all application rates compared to equivalent
treatments applied in late June. Measurements of penetration of the Trichoderma sp. into
the canes and vigour of subsequent fungal growth out into culture were not significantly
different between the three concentrations of the treatments applied. The penetration
distances down the canes of the fungus were generally similar to those assessed in the
previous trial, as were the vigour scores for mycelial growth out of the canes. TABLE 2: Trichoderma incidence (%), penetration (distance down cane from the pruning wound, mm) and vigour (growth radius out from cane into culture, mm) after treatment of pruned grapevine canes with different concentrations of VinevaxTM in August. DISCUSSION
This is the first report of the efficacy of penetration of Trichoderma spp. into grape
wood through wounds at different times after pruning. All other studies (Di Marco et al.
2004; John et al. 2004) have applied the treatments immediately after cutting. Moreover,
Munkvold & Marois (1994) showed that pruning wounds remain susceptible to infection
from E. lata for 4 weeks and European experiences on wound susceptibility suggests that
vine pruning be delayed as late a possible in the winter (Di Marco et al. 2004).
The two major differences between these present trials were the time of the season
when the treatments were applied and the concentration of VinevaxTM applied to the
cut surfaces. The ability to apply sufficient inoculum on to the cut surfaces may be
compromised by ground speed at application as well as the application of a blanket spray
treatment to the whole vine compared to targeted, hand-applied treatments.
With a balance required between maximising the colonisation of the pruned vines and
protection against air-borne and possibly even water-borne vine pathogen inoculum, it
would appear from the results in this study that treating wounds with VinevaxTM should
be carried out at the end of the day. A balance between the completion of pruning
and the application of the wound protection on the same day must be worked out by
operators within the vineyard, especially when considering the limited daylight hours
available at this time of the year. The difference in time of application during the season
2006 New Zealand Plant Protection Society (Inc.) www.nzpps.org Refer to http://www.nzpps.org/terms_of_use.htmlViticulture
of the two trials may have been a contributing factor to the variation in results. Thus,
it is planned to repeat the trails both early and late in the season using both spur and
We thank Penny Turpin and Winna Harvey for their valuable assistance in carrying
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