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Melo, M.J.; Machado, M.H.; Moniz, M. de F.; Bragança, M.H.; Sousa Santos, M.N.
Resistance patterns of Cryptomeria japonica to Armillaria mellea in S. Miguel Island—
Azores. In Proceedings of the Poster 4J Congresso da Sociedade Portuguesa de
Fitopatologia, Universidade do Algarve, Faro, Portugal, 4–6 February 2004.
Resistance patterns of Cryptomeria japonica to Armillaria mellea in S.
Miguel Island - Azores
Maria João Melo (1), Maria Helena Machado (1), Maria Helena Bragança (1), Maria Natércia
Sousa Santos (1), Maria de Fátima Moniz (1), Sónia Feio (2)
(1) Estação Florestal Nacional, Quinta do Marquês, 2780 – 159 Oeiras, e-mail:
helena.machado@efn.com.pt
(2) Instituto Nacional de Engenharia e Tecnologia Industrial, Estrada ao Paço do Lumiar,
1690-038 Lisboa, e-mail: sonia.feio@ineti.pt
Abstract
Armillaria root disease has been considered, since several decades, the cause of heavy damages and
economic losses in S. Miguel Cryptomeria japonica (L.F.) D. Don. stands. As such, several studies have
been done having as main goal to improve the knowledge on disease distribution, incidence and host-
pathogen relationships. Recently, new attempts to assess different pathogenicity levels on C. japonica
varieties were made. The results achieved led to the following conclusions; remarkable significative
differences on tree susceptibility were found in accordance with tree heartwood colour varieties; an efficient
disease control may be achieved, at long term, by maintenance of health nursery conditions and progressive
replacement of the rose heartwood tree variety (the more frequent in S. Miguel C. japonica stands) by dark
brown heartwood variety.
Introduction
Since the classic work of Robert Hartig in 1874, Armillaria root disease has been the object of an immense
collective effort, mainly on taxonomy, physiology and pathogenic behaviour. In spite of the work developed,
the contradictory observations and claims, for over a century, put in doubt the pathogenicity of Armillaria
mellea (Vahl. ex Fr.) Kummer. However, after the work developed by Korhonen (1978), it was verified that
A. mellea don’t constitute a unique species. In fact, under the same designation, were included several
Armillaria species with different pathogenecity levels. In Portugal continental A. mellea is very frequent in
forest, orchards and parks, but its role on mortality and production losses is not clarified as it is also verified
worldwide, probably due to the difficulties involved and efforts required (Hood et al. 1991). However, the
observations carried out, since the middle of the last century, in the Cryptomeria japonica (L.F.) D. Don.
stands situated in S. Miguel Island – Azores, makes clear that A. mellea is widely scattered all over the main
representative C. japonica stands in association with high rates of tree decay and mortality (Azevedo, 1960).
On the strength of ecological and economic importance of C. japonica, several surveys were done (Azevedo,
1958, 1960) having as main goal to evaluate sanitary status of nurseries and stands. The same author refers
that among the 36 stands prospected, A. mellea was detected in 30 of them, being the more affected stands
situated in north, southwest, northwest and centre of the island. In these stands, the percentage of the trees
attaint by the fungus, ranges from 60 to 90. Several attempts were also done (Azevedo, 1970-1971) to
determine host varieties-pathogen relationships but results achieved are not conclusive. This paper is
concerned with recent work developed focusing the determination of tree heartwood varieties resistance.
Material and methods
Inoculation tests: A. mellea - C. japonica
The assay was carried out in a greenhouse situated in “Direcção Geral de Serviços de Protecção de Culturas
de S. Miguel”. The seedlings were obtained by germination of seeds collected from C. japonica varieties
having different heartwood colours – rose (RH), brown (BH) and dark brown (DBH). Three groups
containing, each group, 90 2-year-old seedlings proceeding from trees with the same heartwood colour, were
grown in pots and distributed in three sub-groups including each one, 30 plants (total number of plants-270).
These sub-groups were disposed at random. The inoculations were done in 15 seedlings, per sub-group, with
mycelium removed from 4-week-old colonies of A. mellea growing in Malt-Agar (Difco) previously isolated
from an infected C. japonica tree. A piece of mycelium (0.25 cm2) was inserted under the collar bark,
through a longitudinal cut (1.0 cm length) posteriorly covered with wet cotton and cellophane paper for 10
days. The remaining 15 seedlings, per sub-group, were inoculated with sterile Malte-Agar as control. The
isolate used was previously confirmed as A. mellea by “pairing test” method (Khoronen, 1978) and PCR-
based restriction fragment length polymorphism (RFLP-PCR) (Bragança, 1999). The temperature and
relative humidity were maintained at 20-24º C and 70-75% respectively. Fungus reisolation was made 8
months after inoculation, by removing aseptically small pieces of tissues at level of inoculation zones. Each
piece was put in a Petri dish containing Malt Agar (Difco) amended with 100ml/l of Penicillin and 100ml/l of
Streptomycin. Both seedling symptoms and fungus signs were recorded at the end of 8 months of the test.
Statistical analysis was done by Fisher PLSD method (5% confidence level).
Evaluation of A. mellea growth. Relative inhibition
The bioassay was performed according to Henricks et al. (1979) and Santos (1987) respectively for the
obtention of the 3 heartwood variety extracts (rose heartwood-RH; brown heartwood-BH; dark brown
heartwood-DBH) and evaluation of fungus growing, by determination of both colony diameter and mycelium
dry weight. The extracts were obtained by dissolution of the 3 sawdust heartwood varieties in Methanol. A
concentration of 10 -1 was obtained by mixing the extracts in Malt Agar (Difco) culture medium. The final
culture medium was distributed in Petri dishes (diameter 9 cm) for solid medium and in Khol flasks for liquid
medium, being the amounts used 20cm3 and 10cm3, respectively. Control cultures containing only the solvent
were similarly prepared. The cultures were kept in a flow chamber during 24 h in order to evaporate the
solvent and inoculated with a plug of mycelium removed from the margin of active growing colonies. The
cultures were incubated, in dark, at 25º C. The evaluation of the diameter growth started 6 days after
incubation in a total of 6 evaluations made at each 4 days, while the evaluation of mycelium dry weight was
done at the end of the assay. A number of 6 and 3 replicates were respectively used to the determination of
diameter growth and mycelium dry weight. The Relative Inhibition (RI) was calculated as follows: RI=(D0-
D) x 100/D0 being D0 the diameter of the control colonies and D the diameter of the colonies treated with the
extracts.
Results and discussion
Inoculation tests: A. mellea - C. japonica
The results achieved (Table 1) confirm that the trees with rose heartwood (RH) are more susceptible than
those with brown (BH) and dark brown heartwood (DBH). Taking into consideration the 3 heartwood
varieties, 93,3% of the RH trees present disease symptoms and fungus signs, dying later, while the BH, DBH
varieties and controls present 57,8%, 0% and 0% respectively. A remarkable resistance of the trees included
in the DBH variety is clearly shown. Previous results obtained by Azevedo (1970-1971) using RH variety
and “other heartwood colours” (BH and DBH were not differentiated by the author) don’t permit to found
clear differences about susceptibility among the 3 varieties. Otherwise, the different methodology used
mainly as it concerns the different origin of the A. mellea isolates and the uncertainness of fungus
identification as A. mellea (sensu stricto) may be an explanation for the different results obtained. In fact, a
particular constraint for fungus specific identification has been observed in S. Miguel - the fungus inability to
produce carpophores under the ecological conditions of the island. Otherwise, at four decades ago, “pairing
tests” and molecular methods were not available.
Table 1. Number of C. japonica plants tested and percentage average (± standard deviation) of plants with
root rot, rizomorphs and mycelial sheets, 8 months after inoculation with A. mellea. Different letters indicate
significant differences at 95% confidence level.
Heartwood
colour
Treatment
Number of
plants
% of plants with
root rot
% of plants with
Rizomorphs
Mycelial sheets
Rose (RH)
Inoculated
3 x 15
93.3 ± 11.5 a
93.3 ± 11.5 a
40.0 ± 13.3 a
Control
3 x 15
0 c
0 c
0 c
Brown (BH)
Inoculated
3 x 15
57.8 ± 10.2 b
57.8 ± 10.2 b
15.6 ± 3.8 b
Control
3 x 15
0 c
0 c
0 c
Dark Brown
(DBH)
Inoculated
3 x 15
0 c
0 c
0 c
Control
3 x 15
0 c
0 c
0 c
The observations carried out, in field, by Azevedo (1966) and also by forestry authorities of the island, permit
to conclude that the RH variety is more frequent and more affected than BH and DBH varieties. This fact
explains the remarkable impact of the disease in this ecosystem. In spite of the biological knowledge increase
since the time of Hartig, the efficiency of control measures, with some exceptions, has not improved very
much, mainly in forestry ecosystems where economic and environmental unfavourable impacts have to be
taken into consideration. However, a management model was proposed by Shaw III et al. (1991) mainly
concerned with inoculum removal and silvicultural treatments. Other integrated measures and
recommendations to reduce disease losses are also pointed out by Hagle & Shaw III (1991) and Azevedo
(1960). In this specific case, the knowledge obtained, allow to conclude that the maintenance of sanitary
health status of nurseries and also the progressive replacement of the RH variety by DBH may be the more
efficient procedures for the recuperation of this ecosystem at long term.
Evaluation of A. mellea growth. Relative inhibition
The results achieved confirm those of inoculation tests. In fact the determination of fungus growing by
colony diameter and mycelium dry weight methods, present similar results (Fig.1A, Fig. 2A). The colonies
on medium containing extract from BDH have a significant decrease of growth when compared with control
and media containing RH and BH extracts. The relative inhibition (Fig.1B, Fig. 2B) makes also clear the
repressive effect of DBH extract. On contrary, for RH and BH extracts, the inhibition values are negative
when compared with control indicating that these extracts include in their composition favourable
substance(s) enhancing fungus growth. New attempts to achieve chemical characterization of fungistatic
compound(s) in DBH extract will be the object of the next attempts.
Figure 1. Colony diameter average - A and relative inhibition (%) - B. Bars represent standard deviation.
Different letters indicate significant differences at 95% confidence level.
Rose heartwood (RH); Brown heartwood (BH); Dark brown heartwood (DBH); Control.
(%)
RH
BH
DBH
-25
0
25
50
75
(cm)
0,00
2,00
4,00
6,00
8,00
RH BH DBH Control
B
a
a
b
a
A
Figure 2. Mycelium dry weight average - A and relative inhibition - B. Bars represent standard deviation.
Different letters indicate significant differences at 95% confidence level.
Rose heartwood (RH); Brown heartwood (BH); Dark brown heartwood (DBH); Control.
Conclusions
The disease is widely scattered in the more representative S. Miguel C. japonica stands presenting high
incidence values mainly for rose heartwood trees which are also the more frequent in all the stands. This fact
associated with climate factors (very favourable to fungus growth) explains the remarkable disease impact.
The results of the field observations previously done and assays carried out in nursery (inoculation tests) and
laboratory (fungus growth inhibition) clearly coincide, allowing to conclude that there are significative
differences on susceptibility levels among tree heartwood C. japonica varieties, mainly between the trees
with rose (susceptible) and dark brown (resistant) heartwood.
On the strength of the ecological and economic constraints to control Armillaria root rot in forestry stands,
the more suitable measures for an efficient disease control, at long term, seems to be the maintenance of
nurseries health status and the progressive replacement of the rose heartwood variety by dark brown
heartwood.
References
Azevedo, N.F.S. 1958.Uma armilariose em Cryptomeria japonica (L.F.) D. Don. Publicações da Direcção
Geral dos Serviços Florestais e Aquícolas, XXV: 59-75.
Azevedo, N.F.S. 1960. O “brocado” dos povoamentos de Cryptomeria japonica D. Don na ilha de S. Miguel.
Estudos e Informação nº 124-D3. Direcção Geral dos Serviços Florestais e Aquícolas, Lisboa.
Azevedo, N.F.S. 1966. Acerca da Armillaria mellea (Vahl. ex Fr.) Kummer. Tese. Instituto Superior de
Agronomia, Lisboa, 129 pp.
Azevedo, N.F.S. 1970-1971. Acerca de Armillaria mellea (Fr.) Kummer. Inoculações experimentais em
Cryptomeria japonica (L.F.) D. Don e Quercus suber L. Boletim da Sociedade Portuguesa de Ciências
Naturais XIII: 39-90.
Bragança, M.H. 1999. Análise Fenética em Armillaria-Pesquiza de Marcadores Bioquímicos e Moleculares
para Diferenciação Inter-específica. Tese de Mestrado. Faculdade de Ciências da Universidade de Lisboa.
83pp.
Hagle, S.K. & Shaw III, C.G. 1991. Avoiding and reducing losses from Armillaria root disease. In: Shaw III,
C.G. & Kile, G.A., eds. Armillaria Root Disease. United States Department of Agriculture, Forest Service,
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(g)
0,00
0,10
0,20
0,30
0,40
0,50
RH BH DBH Control
(%)
RH
BH
DBH
-100
-75
-50
-25
0
25
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75
100
a
b
c
b
A
B
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Botaniker und Forstmanner. Berlin, Springer, 127 pp. cit. by Shaw III & Kile, 1991.
Henricks, M.L.; Ekman, R.; Von Weissenberg, K. 1979. Bioassay of some resin and fatty acids with Fomes
annosus. Acta Acad. Aboensis 39B: 1-7.
Hood, I.A.; Redfern, D.B.; Kile, G.A. 1991. Armillaria in planted hosts. In: Shaw III, C.G. & Kile, G.A., eds.
Armillaria Root Disease. United States Department of Agriculture, Forest Service, Agriculture Handbook nº
691: 122-149. Chapter 9.
Korhonen, K. 1978. Interfertility and clonal size in the Armillariella mellea complex. Karstenia 18: 31-42.
Santos, M.N.S. 1987. Contribuição para o estudo de Discosporium populeum (Sacc.) Sutton (syn. Dothichiza
populea Sacc. et Briard). Tese. Instituto Nacional de Investigação Agrária, Lisboa, 160 pp.
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Service, Agriculture Handbook nº 691.
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root disease. In: Shaw III, C.G. & Kile, G.A., eds., Armillaria Root Disease. United States Department of
Agriculture, Forest Service, Agriculture Handbook nº 691: 150-156. Chapter 10.