Modern aspects of biological control of Fomes and Armillaria1

Biological methods can help to control root-infecting fungi such as Fomes annosus and Armillaria mellea. For example, inoculation of pine stumps with the fungus Peniophora gigantea is widely used against F.annosus in Britain. Experiments arc in progress to determine whether similar methods can be used for stumps of other conifers. The main sources of infection by Armillaria are stumps of broad-leaved trees, which are often treated chemically to prevent regrowth. Some of these treatments promote colonization by saprophytic fungi that compete well with Armillaria. In experiments, direct inoculation of stumps combined with chemical treatment has given encouraging results.     

Inoculation of pine stumps with Peniophora gigantea by chainsaw felling

By adding oidia of Peniophora gigantea to the lubricating oil of a chainsaw, stumps were automatically inoculated as the trees were felled. An experiment on Pinus sylvestris showed that direct chainsaw inoculation gave comparable results in terms of abundance of P. gigantea and control of Fames annosus, as conventional inoculation of stumps after felling.     

Dry weight loss of wood after the inoculation of Scots pine stumps with Phlebiopsis gigantea

Artificial inoculation of stumps with Phlebiopsis gigantea (preparation‘PgIBL’) against Heterobasidion annosum in Scots pine (Pinus sylvestris) stands on former agricultural lands in Poland is being performed throughout the year. The practical efficiency of the biological control appears to be influenced by the moisture content in stumps and roots. In experiment 1, dry weight loss of wood 3 months and 6 months after P. gigantea inoculation in laboratory was investigated in the stumps after salvage cutting (felling of dead and dying trees) and after thinning (routine cutting of trees) and compared with the decay of the artificially inoculated stumps under field conditions. It was found that 6 months after inoculation the dry weight loss of the samples was about 3%, 17% and 22%, respectively. In experiment 2, the decay of wood from horizontal roots collected after thinning, inoculated with P. gigantea in laboratory, was evaluated. Three months after the inoculation, the loss of dry weight wood was about 22–52%, depending on initial moisture of the roots.     

Evaluation of the biological control agent Rotstop in controlling the infection of spruce and pine stumps by Heterobasidion in Latvia

The biological control agent Rotstop^® composed of a suspension of spores of Phlebiopsis gigantea (Fr.) Jül. is widely used for protecting conifer stumps from aerial infection by Heterobasidion species. The efficacy of Rotstop application on Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) stumps was determined in several locations and at different seasons in Latvia. Mean efficacy in controlling natural infection by Heterobasidion spp. in spruce stumps was 64%, calculated on the basis of number of infected stumps, and 89%, calculated on the basis of area of infected wood on sample discs cut from the stumps. Corresponding proportions for pine were 82% and 95%. The results show that Rotstop can be successfully used for stump treatment in Latvia, although improved efficacy is desirable, particularly in spruce. A Latvian isolate of P. gigantea, selected from numerous isolates in preliminary tests, was included in one experiment and was shown to be as effective as the Rotstop isolate. In untreated spruce stumps Heterobasidion spp. and P. gigantea were present in the same stump three times more frequently than in untreated pine stumps. Heterobasidion spp. infection in untreated spruce stumps was low when P. gigantea covered more than 10% of stump dissection.     

Persistence of a biocontrol strain of Phlebiopsis gigantea in conifer stumps and its effects on within‐species genetic diversity

Fungal isolations and genetic fingerprinting were used to determine whether Phlebiopsis gigantea stump treatment against Heterobasidion annosum sl. using a single genotype (Rotstop) would affect the genetic diversity of P. gigantea populations. The survival time of P. gigantea was longer in Norway spruce (Picea abies) stumps compared to Scots pine (Pinus sylvestris) as no isolates were obtained from pine stumps 6 years after treatment, whereas in about half of the spruce stumps the fungus was still present. The usage of Rotstop did not seem to increase the occurrence of the fungus 5 years after the treatment in fresh (1‐year‐old) untreated stumps within the same forest stands. All the isolates from the 6‐year‐old treated spruce stumps were identical in genotype with the Rotstop‐strain, whereas all isolates from the fresh untreated spruce and pine stumps differed from it. Within the treated pine stand, the biocontrol usage seemed to have caused a slight reduction in genetic markers not related to Rotstop, but there were no statistically significant differences between the marker frequencies and the local natural population. Thus, Rotstop is not likely to cause any immediate threat to the genetic diversity of P. gigantea.     

An Experiment to Control Fomes annosus in Second Rotation Pine Crops

Stump removal was found to singnificantly reduce killing byFomes annosus in second rotation crops of pine. Poisoning andgirdling of trees prior to felling and also stump poisoningfailed to control the disease. In this experiment no significantdifferences were detected in the susceptibility of Scots andCorsican pine to attack. Increasing periods of delay beforereplanting resulted in less mortality. An equation is presented,based on factors of the first rotation crop which may be usedto predict losses likely to occur when a site is replanted.Root attack by F. annosus results in reduced growth and yieldas well as mortality. The results from the experiment are discussed in trems of themangement of pine crops attacted by F. annosus in Theford Chase.     

The effect of Phlebiopsis gigantea and urea stump treatment against spore infection of Heterobasidion spp. on hybrid larch (Larix × eurolepis) in southern Sweden

The efficacy of stump treatment with 40% urea solution and spore suspension of Phlebiopsis gigantea against primary infection by Heterobasidion spp. on hybrid larch (Larix × eurolepis) was tested in two field studies in southern Sweden. In the first study, stumps treated with urea or P. gigantea were sampled by cutting a cross‐section disc after 3 months following exposure to natural conditions, and in the second study, stumps treated with only P. gigantea were sampled after 2 months. Spore traps made from hybrid larch, Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) were used to estimate the abundance of ambient spores of Heterobasidion spp. in the second study. Urea significantly reduced infection frequency of Heterobasidion spp. compared to the control. Phlebiopsis gigantea was less effective at reducing infection frequency in the first study than in the second study. Infections were caused by both H. parviporum and H. annosum sensu stricto. The amount of H. annosum infection in proportion to Heterobasidion spp. was significantly lower on the stumps treated with P. gigantea compared to the control; however, the proportion of H. parviporum on the treated stumps was not higher than the control in the first study. Spore traps made of Scots pine had a significantly higher frequency of infection than Norway spruce and hybrid larch. Only the spore traps made of hybrid larch showed significant correlation with the control stumps in terms of relative infected area. Conclusively, it seems prudent to protect hybrid larch stumps from primary infection by Heterobasidion spp., and both urea and P. gigantea can be recommended as stump treatment agents on hybrid larch, even if urea seemed to present more stable results.     

Growth of Phlebiopsis gigantea in wood of seven conifer species

Heterobasidion parviporum and Heterobasidion annosum are widely distributed root‐rot fungi that infect conifers throughout Europe. Infection of conifer stumps by spores of these pathogens can be controlled by treating fresh stumps with a competing non‐pathogenic fungus, Phlebiopsis gigantea. In this study, growth of three Latvian strains of P. gigantea and the biological control agent ‘Rotstop’ strain was evaluated in stem pieces of Norway spruce, Scots pine, lodgepole pine, Douglas‐fir, Weymouth pine, Siberian larch and Sitka spruce. The growth rates of one H. parviporum and one H. annosum isolate were also measured in the same stem pieces. The growth rate of P. gigantea varied greatly in wood of different conifer species. It was higher in the three pine species, lower in Norway spruce and lowest in Sitka spruce and Siberian larch, and in Douglas‐fir, this fungus did not grow. The largest area of wood occupied by P. gigantea was in lodgepole pine. Growth of Latvian isolates of P. gigantea in the wood of Pinus and Picea species was comparable to that of the Rotstop isolate. Consequently, stump treatment with local P. gigantea isolates should be recommended. However, our results suggest that Douglas‐fir stump treatment against Heterobasidion by P. gigantea may be ineffective and other stump treatment methods should be considered.     

Biological control of Heterobasidion annosum on Pinus pinea by Phlebiopsis gigantea

The efficacy of a native isolate of Phlebiopsis gigantea in protecting the stumps of Pinus pinea against spore infection by Heterobasidion annosum s.str. was investigated. In preliminary tests carried out in stem pieces of P. pinea in confined environment, the efficacy of the isolate was compared with Rotstop^®, the commercial formulation prepared from a north European P. gigantea strain. Both showed a fully protective effect against artificial inoculation of H. annosum. Moreover, in stump treatment experiments carried out in a coastal P. pinea forest close to Rome, the native P. gigantea was effective against heavy airborne inoculum of H. annosum. Some natural infection by P. gigantea occurred in the stumps, but it was unable to control the pathogen. Random amplified microsatellite analysis allowed to recognize the presence of different P. gigantea strains in the forest.     

Stimulatory effect of ammonium lignosulfonate on germination and growth of Phlebiopsis gigantea spores

Ammonium lignosulfonate (ALS) was found to stimulate the germination and growth of Phlebiopsis gigantea. When incubated in 1% and 2.5% solutions germination rates were higher and germ tubes longer at all temperatures tested when compared to water controls. When ALS was used to inoculate P. gigantea into red pine stumps the spores had germinated and hyphae were easily observed after 16 h but no germination had occurred in the stumps treated with oidia in water.     

Relationship between stump treatment coverage using the biological control product PG Suspension,and control of Heterobasidion annosum on Corsican pine,Pinus nigra ssp. laricio

The relationship between the proportion of the stump surface covered by the biological stump treatment agent PG Suspension, containing Phlebiopsis gigantea and its efficacy against the pathogen Heterobasidion annosum sensu stricto was studied during a first thinning of Corsican pine (Pinus nigra ssp. laricio) in Thetford Forest, UK. PG Suspension was manually applied to 100%, 75%, 50% or 0% of the surface of 150 stumps. Spores of H. annosum were inoculated onto 75 of the stumps, and the remaining stumps exposed to natural airborne spore deposition. The relationship between coverage and efficacy was found to be quantitative. Covering all the stump surface with PG Suspension completely excluded the pathogen, whereas stumps not treated with PG Suspension (the 0% treatment) became infected with H. annosum. Partial (75%) PG Suspension coverage resulted in the pathogen colonizing 40% of stumps following artificial inoculation with H. annosum, and just 7% of stumps exposed to ambient H. annosum spore infection. Decreasing levels of coverage allowed increasing areas of the stump surface to be colonized by H. annosum. Some small gaps in coverage were closed by lateral growth of P. gigantea, but it is recommended that operators aim for full stump coverage to give complete protection against H. annosum.     

Effect of Thinning and Phlebiopsis gigantea Stump Treatment on the Growth of Heterobasidion parviporum Inoculated in Picea abies

The spread of Heterobasidion parviporum Niemelä & Korhonen in roots of Norway spruce was studied in three unthinned first rotation stands of Norway spruce [Picea abies (L.) Karst.] on former agricultural land in south-western Sweden. Heterobasidion parviporum was inoculated at stump height into the trunk of 135 standing trees in a randomized block design. One year after inoculation, two-thirds of the trees were thinned out and one-third was left standing. Half of the stumps left by thinning were treated with spores of Phlebiopsis gigantea (Fr.) Jül and half were left untreated. The spread of H. parviporum was examined both 3 and 5 yrs after inoculation. The rate of spread of H. parviporum and the proportion of infected roots were found to be significantly higher in the root systems of the stumps than in those of the standing trees. Treatment with P. gigantea had no significant effect on the development of H. parviporum in the stumps. There was a tendency 5 yrs after inoculation, however, for a lower proportion of H. parviporum-infected roots in the stumps treated with P. gigantea than in the untreated stumps. In conclusion, thinning of infected Norway spruce was found to increase the rate of spread of H. parviporum in the root systems of the infected trees, which could increase the risk of a rapid build-up of infection in the remaining stand.     

Fomes annosus (Fr.) Cke. and other Root-rotting Fungi in Conifers on ex-Hardwood Sites

The development of root rot and butt rot caused by Fomes annosusin conifer crops planted on ex-hardwood ground is slower thanthat in second-rotation conifer plantations. This is apparentlydue to competition from other fungi, notably Artnillaria mellea.Excavations on ex-hardwood sites showed that F. annosus waspresent in conifer thinning stumps, and that transference ofinfection occurred, causing butt rot. The investigations confirmedthe need for stump protection of all conifer crops planted onex-hardwood sites. Short accounts are given of the activityof A. mellea and Polyporus schweinitzii, the other two importantroot-rotting fungi on these sites.     

Tolerance of Fomes annosus isolates to pine oleoresins and pinosylvins

Ten Fomes annosus isolates from pine and ten from non-pine-hosts were subjected to the volatile components of pine oleoresin and to various concentrations of pinosylvin (PS) and pinosylvin monomethyl ether (PSM). Both the volatiles and the PS and PMS significantly reduced growth rate compared with controls. However, no detectable difference existed between the two groups of F. annosus isolates in their reactions to the substances tested.     

Armillaria novae‐zelandiae and other basidiomycete wood decay fungi in New Zealand Pinus radiata thinning stumps

Two studies were undertaken in a young New Zealand Pinus radiata stand to find a biological control agent for armillaria root disease caused by Armillaria novae‐zelandiae. Fresh thinning stumps were inoculated with saprophytic basidiomycete species as wooden dowel cultures or as homogenized aqueous mycelial suspensions, while all stumps were also inoculated with dowel cultures of A. novae‐zelandiae. After a period of between one and two years, no differences were found among test fungi in the percentage of Armillaria species isolated from stumps. However, inoculation of Stereum sanguinolentum as homogenate, and of Phlebiopsis gigantea, Sistotrema brinkmannii, Hypholoma acutum and Rigidoporus concrescens as dowel cultures, significantly increased their isolation incidence in comparison with stumps not treated by these species. Phlebiopsis gigantea, Resinicium bicolor and R. concrescens were also isolated with greater frequency nearer to their dowel inoculation points. Results suggest that P. gigantea and S. sanguinolentum should be included in further testing. However, a future trial is likely to be more effective if evaluation is directed towards the portion of the stump below the soil surface, particularly the root system.     

Comparison of five strains of Phlebiopsis gigantea and two Trichoderma formulations for treatment against natural Heterobasidion spore infections on Norway spruce stumps

In an experiment established on three Norway spruce sites in southern Sweden, the ability of five strains of Phlebiopsis gigantea, including the commercial strain Rotstop®, and two Trichoderma formulations to control natural Heterobasidion spp. spore infections was compared. At each site 160 trees were felled, and the resulting stumps were treated with spore suspensions of the seven fungal preparations. Twenty stumps at each site were left untreated as control stumps. When sampled 9 months after treatment, two of the P. gigantea strains (1984 and 1985) were the most effective at preventing infection by Heterobasidion spp. The other three P. gigantea strains were less effective, and two Trichoderma formulations did not significantly reduce Heterobasidion spp. infections.     

Heterobasidion annosum infection of Picea abies following manual or mechanized stump treatment

Heterobasidion annosum (Fr.) Bref. s. l. colonization following thinning was studied in 1246 stumps of Picea abies (L.) Karst. trees from 14 previously unthinned stands in Sweden. Treatments included mechanized and manual application of (1) 35% urea solution, (2) Phlebiopsis gigantea (Fr.) Jül. spores, and (3) 5% solution of disodium octaborate tetrahydrate (DOT), and untreated stumps, cut in the summer and winter. Compared with untreated stumps cut in the summer, all stump treatments and winter cuttings significantly reduced the colonized stump area 6–7 weeks after thinning by 88–99%. Mechanized stump treatment provided as good protection as manual treatment against H. annosum infections. The probability of spore infection (p _ij ) was reduced by 53–83% in mechanized treatment and 79–98% in manual treatment compared with untreated summer thinning. In terms of p _ij , urea had significantly higher control efficacy than P. gigantea and manual treatment performed better than mechanized treatment.     

A Study of Losses in Scots Pine caused by Fomes annosus

Data are presented from unreplicated Scots pine {Pinus sylvestrisL.) sample plots in Thetford Forest which have been differentiallyattacked by Fomes annosus (Fr.) Cooke. Volume production ofseverely infested crops differs markedly from that forecastby management tables. The volume of dead trees may not representthe total loss of volume in these plots as there appears alsoto be a reduction in the growth of the remaining live trees.These observations may have considerable significance in forecastingfuture volume production from stands attacked by F. annosusand also in deciding the optimum time for their replacement.     

Untersuchungen zur biologischen Bekämpfung von Heterobasidion annosum an Fichte (Picea abies) mit antagonistischen Pilzen II. Interaktionstests auf Holz

Investigations on biological control of Heterobasidion annosum in Norway spruce with antagonistic fungi. II. Interaction experiments in wood . Seventeen fungal species were examined for antagonism against H. annosum in wood. After inoculation of stem sections and stumps by conidia and dowels, the distribution patterns of the mycelia within the wood were recorded. In spruce-wood antagonism was shown only by Hypholoma capnoides, Bjcrkandera adusta, Resinicium bicolor and Trichoderma spp. near the site of inoculation. As the distance from this site increased H. annosum became more dominant. Inoculations by dowels yielded more infections than inoculations by spores. The patterns of myeelial distribution within the wood were similar in stem sections and in stumps. Spruce stumps were colonized naturally mainly by Resinicium bicolor, Armillaria mellea s. I. and Nectria fuckeliana. In stem sections of Pinus sylvestris, however, Phlebiopsis gigantea displaced H. annosum effectively.     

Inhibitory effect of Trichoderma viride Pers. ex Fr. filtrates on Fomes annosus (Fr.) Cke. In relation to some carbon sources

The mycelial yield of Trichoderma viride on a number of carbon sources and the inhibitory effect of its filtrates on Fomes annosus were investigated. d-Xylose and d-glucose as carbon sources in the media gave the highest degree of inhibition when their filtrates were directly tested against F. annosus mycelium. The protection of pine wood blocks with filtrates was most effective when these were derived from cellulose and d-xylose.