BACKGROUND
tropical and subtropical forests once covered large areas of central- and south america.
due to high rates of deforestation up to the 80ies of the last century, and also wildfires, large areas are now grasslands or campos  <cit> , or are used for agricultural purposes .
species of the coniferous genus araucaria are important members of tropical and subtropical forests of the southern hemisphere  <cit> .
among them, brazil pine  was one of the most important species, economically and ecologically  <cit> , occurring in mountain areas  of southern brazil, and dominated the forest vegetation  <cit> .
due to severe clear cutting and fires, native araucaria forests today occupy only 1% of the original area occupied  <cit> .
brazil pine is thus an endangered species  <cit> .
recent investigations, however, show that under undisturbed conditions forest land starts to invade the grasslands again  <cit> .
araucariaceae represent very ancient gymnosperms and are also called “living fossils”.
according to largely missing literature on this subject, these trees are obviously not very sensitive to fungal pathogens in comparison to conifers of the northern hemisphere.
in the latter, root-rot inducing species such as heterobasidion spec.
cause considerable losses in wood production  <cit> .
there is, however, a recent report on root and crown rot in a. angustifolia, caused by phytophthora cinnamomi <cit> , and most recently, dalmas and astarita  detected a fungal pathogen in a. angustifolia seedlings, which severely inhibited seedling development.
with regard to biocontrol, streptomycetes, which are an important part of bacterial communities of the rhizosphere, have attracted special attention.
streptomycetes produce and release a wide variety of secondary metabolites.
approximately  <dig>  out of  <dig>  biologically active secondary metabolites, such as antibiotics, have been characterized from streptomycetes  <cit> .
when released to the soil, these may contribute to biocontrol, including the induction of systemic resistance in streptomycetes-colonised plants  <cit> .
in studies with spruce seedlings, it could be shown that streptomycetes from the rhizosphere of a spruce stand could systemically improve resistance of seedlings against fungal infection  <cit> .
it was the aim of this study to identify the newly isolated fungal pathogen of a. angustifolia seeds and screen for rhizosphere streptomycetes which, upon germination on ground, can affect the growth of this pathogen.
furthermore, we present a list of exudate compounds produced by the fungus-inhibiting bacteria in single culture, and alterations due to the co-culture with the fungal pathogen.
RESULTS
the pathogenic fungus on a. angustifolia seedlings: effects and identification
after 50 days of germination, about 30% of araucaria seedlings were infected by a fungus that promoted the death of the cotyledons and interrupted the connection between the seedling and the megagametophyte .
of these, about 50% died, and the surviving ones showed delay in plant development.
after 150 days,  <dig> % of surviving plants with retarded development were dead.
the cause for delayed development or seedling death might be attributed to the early interruption in the carbon and nutrients transfer from the megagametophyte to the embryonic tissues.
electron microscopy analyses showed the presence of high amounts of starch grains in the megagametophyte of infected seedlings , compared with the non-infected tissue .
the natural infection of the a. angustifolia seeds by the fungus might have happened during cone maturation and before seed dispersion.
the fungus infected specifically the megagametophyte tissue and promoted necrosis of the seed-enclosed region, and the cotyledons, after their emergence.
the first visible symptoms were the decay of the cotyledons and seed browning.
in this species, the cotyledons act as a haustorial organ by transferring the reserves from the megagametophyte to the embryonic axis  <cit> , supporting the seedling growth until about  <dig> to 120 days  <cit> .
the early cotyledon interruption leading to seedling death or delayed plant development, significantly reduced the chances for seedling establishment.
its sequencing of the fungal isolate with the primer pairs its <dig> and its <dig>  yielded the highest homologies  with neofusicoccum parvum/n.
ribis and botryosphaeria parva, all members of the botryosphaeriaceae.
this is due to the fact that neofusicoccum parvum is the anamorph of botryosphaeria parva <cit> .
n. parvum and n. ribis were originally considered to be part of the botryosphaeria dothidea complex  <cit> .
currently, these two neofusicoccum species, together with three cryptic species isolated from syzygium cordatum in south africa, are regarded as forming a unique group, named the n. parvum/n.
ribis complex  <cit> .
however, only neofusicoccum parvum has been frequently associated with brown streaking and necrosis of wood  <cit> .
based on genomic markers, pavlic et al.
<cit>  identified five groups, n. parvum, n. ribis, and three distinct lineages within the np/nr complex.
sequences of its , ef-1a , bt , botf <dig> , or rpb <dig>  of the unknown fungi, did not contain one of the snps characteristic for n. ribis or the members of the three lineages n. sp r <dig>  n. sp r <dig>  or n. sp r <dig>  alignment of the its-sequences revealed one indel at position  <dig> to n. ribis  and one snp at position  <dig> to n. parvum .
based on these data and a report about the identification of n. parvum on a. heterophylla <cit>  we suggest this fungus is n. parvum.
this fungus has been reported in both brazil and australia.
electron microscopy of fungal hyphae strongly supports the sequence data.
figure  <dig> shows septa with simple pores having more or less rounded lips.
the pores are associated with woronin-bodies which identifies the fungus as ascomycete, belonging to the subphylum pezizomycotina  <cit> .
fungi of botryosphaeriaceae occur in a wide diversity of plants and can act in different ways, as primary or opportunistic pathogens, but also as endophytes or saprobes  <cit> .
since such fungi have also been reported to affect araucariaceae, such as the recently discovered wollemia nobilis in australia, as well as araucaria spec.
in new zealand  <cit> , biocontrol properties of australian streptomycetes are not only of local interest.
rhizosphere streptomycetes with biocontrol potential and their exudates
we thus screened streptomycete isolates from australian araucaria stands for potential inhibitors of fungal growth.
as bacterial populations differ between bulk soil and root surface, we tried to isolate bacteria from both sources .
co-culture experiments showed different degrees of growth inhibition .
most effective isolates were m <dig>  m <dig>  m <dig>  mw <dig>  mw <dig> and mw <dig>  sequence analysis of 16s rdna demonstrated that these isolates were streptomycetes.
16s ribosomal rna gene homologies  were with streptomyces albulus , streptomyces chattanoogensis  and streptomyces sp.
ac <dig>  or streptomyces celluloflavus .
from single cultures of bacterial isolates and fungus/bacteria co-cultures on agar,  <dig> different compounds could be identified by comparing the hplc-ms profiles of the respective agar extracts with an in-house hplc-uv–vis database .
the mix of the different exudates was to some degree isolate-specific.
multi dimensional statistical  data analysis illustrates which individual cultures and co-cultures form clusters, and which cultures could be considered similar to one another, on the basis of patterns and combinations due to the presence or absence of exudate compounds.
this approach indicates that the inhibition of the fungus in co-culture  was dependent on the presence of compounds of two groups .
these are group  <dig>  made up by compounds  <dig>   <dig>   <dig> and sometimes  <dig> , and group  <dig>  consisting of compounds  <dig>   <dig>  and  <dig> , each enclosed by circles.
group  <dig> consists of a ß-carboline alkaloid usually extracted from actinomycetes , containing an indole tricyclic ring and is cytotoxic, anti-microbial and an enzyme inhibitor  <cit> .
the other three metabolites in this group are polyene macrolide antibiotics, containing a lactose ring and act against ergosterol of fungal membranes.
filipin is more toxic than lagosin and all three cause excess leakage of k  <cit> .
group  <dig> consist of a peptide antibiotic  that affects glycolytic and lipolytic proteins, and inhibits cell wall formation  <cit> .
the other two compounds  are auxins or auxin antagonists  and may affect many aspects of plant growth and development  <cit> .
compounds  <dig> and  <dig> were generally not released or present from single cultures of either bacteria or fungus, and this is consistent with their roles more directly in plants.
two other well separated metabolites are worth mentioning .
thiolutin  is a well studied broad spectrum indole alkaloid which inhibits energy metabolism, rna synthesis , glucose metabolism and carbon use  <cit> .
n-hydroxy phenyl acetic acid methyl ester is a derivative of indole propionic acid and is a weak alkaloid and anti-microbial compound, acting mainly against gram-negative bacteria  <cit> .
most effective in the inhibition of fungal growth are combinations and the presence of compounds belonging to both group  <dig> and group  <dig>  however, not all metabolites included in these groups are apparently necessary for inhibition.
in co-culture, substances can result from both organisms.
m, isolates from rhizosphere soil; mw, isolates from the surface of araucaria roots.
we could not test the effects of single compounds or combinations thereof, as they are not commercially available.
they only can be obtained from preparative batch cultures.
we have done this before  <cit> , but due to the considerable necessary efforts, this could not be done for the present investigation.
association statistics of the streptomycete isolates and their inhibitory effects on n. parvum revealed that under co-culture, the strong inhibitory bm  and bmw groups  were even more widely separated.
this indicates that the co-cultures showing the highest degree of inhibition were not only different from one another but also very different from the rest of the non-inhibiting cultures with regard to their exudates profiles.
exudates released from the streptomyces isolate m <dig> and n. parvum in single culture and after co-culture were characterized by hplc in more detail .
interestingly, the two identified bacterial peaks  had decreased in amounts in the presence of the fungus.
as detailed before, the macrolide antibiotics are active against yeasts, molds and filamentous fungi, and can cause membrane distortions and leakage of k  <cit> .
the decline in amounts indicates that the fungus also responds to the streptomyces, possibly by taking up these antibiotics which then affect fungal metabolism.
on the other hand, the fungus does not release many compounds into the agar, at least not such ones with low polarity which can be identified by reverse phase hplc.
in recent studies we could show that certain streptomycete isolates can completely abolish disease development caused by the infection of spruce seedlings with the root pathogenic fungi armillaria spec., and heterobasidion spec.
<cit> .
this effect could be attributed to an antibiotic, isolated from the streptomycete  <cit> .
the present study confirms the biocontrol function of many soil bacteria, and especially of streptomycetes.
it also shows that combinations of exudates are obviously more relevant than the application of single compounds.
although the investigation of effector combinations is only a very little step towards the understanding of microbe interactions in the complex rhizosphere.
in ongoing experiments we will try to find out whether the co-culture effects can be simulated by the addition of these compounds , and whether the infection of araucaria seedlings by the fungus can be prevented by co-culture with the respective streoptomycete isolates.
in addition, we have started to screen a range of streptomcete isolates obtained from brazilian araucaria angustifolia stands for their biocontrol function.
for application, spores of efficient bacteria could then be added to a. angustifolia seeds to counteract n. parvum infection.
CONCLUSIONS
streptomycetes from the rhizosphere of araucariaceae produce exudates which can suppress the growth of pathogenic fungi in their seeds.
the focus of this contribution is on the effect of bacteria from australian sources on a brazilian tree species .
however, our most recent studies show that the potential biocontrol properties of brazilian rhizosphere bacteria are very similar to those of australian isolates.
thus, the bacterial impact is not restricted to the respective source of bacteria, or bacteria/species of araucariaceae.

 tropical and subtropical forests once covered large areas of central- and south america. due to high rates of deforestation up to the 80ies of the last century, and also wildfires, large areas are now grasslands or campos, or are used for agricultural purposes. species of the coniferous genus araucaria are important members of tropical and subtropical forests of the southern hemisphere. among them, brazil pine was one of the most important species, economically and ecologically, occurring in mountain areas of southern brazil, and dominated the forest vegetation. due to severe clear cutting and fires, native araucaria forests today occupy only 1% of the original area occupied. brazil pine is thus an endangered species. recent investigations, however, show that under undisturbed conditions forest land starts to invade the grasslands again. araucariaceae represent very ancient gymnosperms and are also called “living fossils”. according to largely missing literature on this subject, these trees are obviously not very sensitive to fungal pathogens in comparison to conifers of the northern hemisphere. in the latter, root-rot inducing species such as heterobasidion spec. cause considerable losses in wood production. there is, however, a recent report on root and crown rot in a. angustifolia, caused by phytophthora cinnamomi, and most recently, dalmas and astarita detected a fungal pathogen in a. angustifolia seedlings, which severely inhibited seedling development. with regard to biocontrol, streptomycetes, which are an important part of bacterial communities of the rhizosphere, have attracted special attention. streptomycetes produce and release a wide variety of secondary metabolites. approximately out of biologically active secondary metabolites, such as antibiotics, have been characterized from streptomycetes. when released to the soil, these may contribute to biocontrol, including the induction of systemic resistance in streptomycetes-colonised plants. in studies with spruce seedlings, it could be shown that streptomycetes from the rhizosphere of a spruce stand could systemically improve resistance of seedlings against fungal infection. it was the aim of this study to identify the newly isolated fungal pathogen of a. angustifolia seeds and screen for rhizosphere streptomycetes which, upon germination on ground, can affect the growth of this pathogen. furthermore, we present a list of exudate compounds produced by the fungus-inhibiting bacteria in single culture, and alterations due to the co-culture with the fungal pathogen.  the pathogenic fungus on a. angustifolia seedlings: effects and identification after 50 days of germination, about 30% of araucaria seedlings were infected by a fungus that promoted the death of the cotyledons and interrupted the connection between the seedling and the megagametophyte. of these, about 50% died, and the surviving ones showed delay in plant development. after 150 days, % of surviving plants with retarded development were dead. the cause for delayed development or seedling death might be attributed to the early interruption in the carbon and nutrients transfer from the megagametophyte to the embryonic tissues. electron microscopy analyses showed the presence of high amounts of starch grains in the megagametophyte of infected seedlings, compared with the non-infected tissue. the natural infection of the a. angustifolia seeds by the fungus might have happened during cone maturation and before seed dispersion. the fungus infected specifically the megagametophyte tissue and promoted necrosis of the seed-enclosed region, and the cotyledons, after their emergence. the first visible symptoms were the decay of the cotyledons and seed browning. in this species, the cotyledons act as a haustorial organ by transferring the reserves from the megagametophyte to the embryonic axis, supporting the seedling growth until about to 120 days. the early cotyledon interruption leading to seedling death or delayed plant development, significantly reduced the chances for seedling establishment. its sequencing of the fungal isolate with the primer pairs its and its yielded the highest homologies with neofusicoccum parvum/n. ribis and botryosphaeria parva, all members of the botryosphaeriaceae. this is due to the fact that neofusicoccum parvum is the anamorph of botryosphaeria parva. n. parvum and n. ribis were originally considered to be part of the botryosphaeria dothidea complex. currently, these two neofusicoccum species, together with three cryptic species isolated from syzygium cordatum in south africa, are regarded as forming a unique group, named the n. parvum/n. ribis complex. however, only neofusicoccum parvum has been frequently associated with brown streaking and necrosis of wood. based on genomic markers, pavlic et al. identified five groups, n. parvum, n. ribis, and three distinct lineages within the np/nr complex. sequences of its, ef-1a, bt, botf, or rpb of the unknown fungi, did not contain one of the snps characteristic for n. ribis or the members of the three lineages n. sp r n. sp r or n. sp r alignment of the its-sequences revealed one indel at position to n. ribis and one snp at position to n. parvum. based on these data and a report about the identification of n. parvum on a. heterophylla we suggest this fungus is n. parvum. this fungus has been reported in both brazil and australia. electron microscopy of fungal hyphae strongly supports the sequence data. figure  shows septa with simple pores having more or less rounded lips. the pores are associated with woronin-bodies which identifies the fungus as ascomycete, belonging to the subphylum pezizomycotina. fungi of botryosphaeriaceae occur in a wide diversity of plants and can act in different ways, as primary or opportunistic pathogens, but also as endophytes or saprobes. since such fungi have also been reported to affect araucariaceae, such as the recently discovered wollemia nobilis in australia, as well as araucaria spec. in new zealand, biocontrol properties of australian streptomycetes are not only of local interest. rhizosphere streptomycetes with biocontrol potential and their exudates we thus screened streptomycete isolates from australian araucaria stands for potential inhibitors of fungal growth. as bacterial populations differ between bulk soil and root surface, we tried to isolate bacteria from both sources. co-culture experiments showed different degrees of growth inhibition. most effective isolates were m m m mw mw and mw sequence analysis of 16s rdna demonstrated that these isolates were streptomycetes. 16s ribosomal rna gene homologies were with streptomyces albulus, streptomyces chattanoogensis and streptomyces sp. ac or streptomyces celluloflavus. from single cultures of bacterial isolates and fungus/bacteria co-cultures on agar, different compounds could be identified by comparing the hplc-ms profiles of the respective agar extracts with an in-house hplc-uv–vis database. the mix of the different exudates was to some degree isolate-specific. multi dimensional statistical data analysis illustrates which individual cultures and co-cultures form clusters, and which cultures could be considered similar to one another, on the basis of patterns and combinations due to the presence or absence of exudate compounds. this approach indicates that the inhibition of the fungus in co-culture was dependent on the presence of compounds of two groups. these are group made up by compounds and sometimes, and group consisting of compounds and, each enclosed by circles. group consists of a ß-carboline alkaloid usually extracted from actinomycetes, containing an indole tricyclic ring and is cytotoxic, anti-microbial and an enzyme inhibitor. the other three metabolites in this group are polyene macrolide antibiotics, containing a lactose ring and act against ergosterol of fungal membranes. filipin is more toxic than lagosin and all three cause excess leakage of k. group consist of a peptide antibiotic that affects glycolytic and lipolytic proteins, and inhibits cell wall formation. the other two compounds are auxins or auxin antagonists and may affect many aspects of plant growth and development. compounds and were generally not released or present from single cultures of either bacteria or fungus, and this is consistent with their roles more directly in plants. two other well separated metabolites are worth mentioning. thiolutin is a well studied broad spectrum indole alkaloid which inhibits energy metabolism, rna synthesis, glucose metabolism and carbon use. n-hydroxy phenyl acetic acid methyl ester is a derivative of indole propionic acid and is a weak alkaloid and anti-microbial compound, acting mainly against gram-negative bacteria. most effective in the inhibition of fungal growth are combinations and the presence of compounds belonging to both group and group however, not all metabolites included in these groups are apparently necessary for inhibition. in co-culture, substances can result from both organisms. m, isolates from rhizosphere soil; mw, isolates from the surface of araucaria roots. we could not test the effects of single compounds or combinations thereof, as they are not commercially available. they only can be obtained from preparative batch cultures. we have done this before, but due to the considerable necessary efforts, this could not be done for the present investigation. association statistics of the streptomycete isolates and their inhibitory effects on n. parvum revealed that under co-culture, the strong inhibitory bm and bmw groups were even more widely separated. this indicates that the co-cultures showing the highest degree of inhibition were not only different from one another but also very different from the rest of the non-inhibiting cultures with regard to their exudates profiles. exudates released from the streptomyces isolate m and n. parvum in single culture and after co-culture were characterized by hplc in more detail. interestingly, the two identified bacterial peaks had decreased in amounts in the presence of the fungus. as detailed before, the macrolide antibiotics are active against yeasts, molds and filamentous fungi, and can cause membrane distortions and leakage of k. the decline in amounts indicates that the fungus also responds to the streptomyces, possibly by taking up these antibiotics which then affect fungal metabolism. on the other hand, the fungus does not release many compounds into the agar, at least not such ones with low polarity which can be identified by reverse phase hplc. in recent studies we could show that certain streptomycete isolates can completely abolish disease development caused by the infection of spruce seedlings with the root pathogenic fungi armillaria spec., and heterobasidion spec.. this effect could be attributed to an antibiotic, isolated from the streptomycete. the present study confirms the biocontrol function of many soil bacteria, and especially of streptomycetes. it also shows that combinations of exudates are obviously more relevant than the application of single compounds. although the investigation of effector combinations is only a very little step towards the understanding of microbe interactions in the complex rhizosphere. in ongoing experiments we will try to find out whether the co-culture effects can be simulated by the addition of these compounds, and whether the infection of araucaria seedlings by the fungus can be prevented by co-culture with the respective streoptomycete isolates. in addition, we have started to screen a range of streptomcete isolates obtained from brazilian araucaria angustifolia stands for their biocontrol function. for application, spores of efficient bacteria could then be added to a. angustifolia seeds to counteract n. parvum infection.  streptomycetes from the rhizosphere of araucariaceae produce exudates which can suppress the growth of pathogenic fungi in their seeds. the focus of this contribution is on the effect of bacteria from australian sources on a brazilian tree species. however, our most recent studies show that the potential biocontrol properties of brazilian rhizosphere bacteria are very similar to those of australian isolates. thus, the bacterial impact is not restricted to the respective source of bacteria, or bacteria/species of araucariaceae. 
molecular data identified the fungus as neofusicoccum parvum. streptomycetes from the rhizosphere of araucariaceae produce exudates which can suppress the development of pathogenic fungi in their seeds. the pathogenic fungus from araucaria angustifolia seeds was identified by morphological markers as belonging to the pezizomycotina. co-cultures on agar of this fungus with certain streptomycete isolates from the rhizosphere, and from the surface of araucaria roots significantly reduced the growth of the fungus.
 araucariaceae are important forest trees of the southern hemisphere. life expectancy of their seedlings can largely be reduced by fungal infections. in this study we have isolated and characterized such a fungus and investigated the potential of streptomyces actinobacteria from the respective rhizosphere to act as antagonists.  the pathogenic fungus from araucaria angustifolia seeds was identified by morphological markers as belonging to the pezizomycotina. molecular data identified the fungus as neofusicoccum parvum. co-cultures on agar of this fungus with certain streptomycete isolates from the rhizosphere, and from the surface of araucaria roots significantly reduced the growth of the fungus. hplc analysis of the agar yielded streptomycete-specific exudate compounds which were partly identified. there were differences in compounds between single and dual cultures.  streptomycetes from the rhizosphere of araucariaceae produce exudates which can suppress the development of pathogenic fungi in their seeds. antibiosisactinomycetesbrazil pinesecondary compoundsanti-fungal compoundsneofusicoccum 