PhenotyPic , itS Phylogenetic and Pathogenic analySeS of Pyllosticta multicorniculata aSSociated with needle blight SymPtomS of balSam fir in canada

Phyllosticta multicorniculata Bissett & Palm causes needle blight on Ab­ ies balsamea  (L.)  Mill.  in  eastern  Canada. The  disease  symptoms  are  similar  to those provoked by Phyllosticta abietis Bissett & Palm on Abies grandis Lindl. in the U.S.A.  Preliminary study, based on rDNA ITS sequence data analysis, revealed that P. multicorniculata formed distinct but closely-related clade with other sister mitosporic Phyllosticta  and  teleomorphic Guignardia  and Botryosphaeria  taxa. However, the phylogenetic position of the fungus is still doubtful which demand further and more advanced molecular investigation. This is the first report of a microconidial stage in P. multicorniculata and, for the first time, description of the developing mucilaginous appendages in macroconidia. This study contributes in disease diagnostics and gives new information on distribution of the disease in Canada.


introdUction
Balsam fir (Abies balsamea (L.) Mill.) is economically a very important coniferous tree in eastern Canada.Among numerous needle diseases of Balsam fir, found in the Sherbrooke region of the province of Quebec (Vu j a n o v i c , S t -A r n a u d , 2001), one unusual coelomycetous fungus was frequently associated with needle blight symptoms.Usually, the fungal attack causes a reddish colour and needle necrosis resulting in mortality of leading branches and severe crown damage.In order to establish an efficient control strategy, the isolation and identification as well as preliminary molecular and pathogenic analyses of this fungal species were completed in laboratories of the Plant Biology Research Institute/Institut de recherche en biologie végétale (IRBV) located within the Montreal Btanical Garden.

material and methodS
For this study, a collection of plant material was carried out according to a methodology reported by Vu j a n o v i ć & S t -A r n a u d (2001).
rDNA region amplification was carried out using the ITS1/ITS 4 primer set (W h i t e et al ., 1990).PCR amplicons were purified for sequencing from agarose gels using the QIAquick gel extraction kit, and sequencing was performed by the Montréal Genomic Centre (Montréal, Qc, Canada).Sequence data were aligned using ClustalW and then imported into PAUP (version 4.0b10) (S w o f o r s , 2003).The resulting tree was exported to TreeView (P a g e , 1996) and rooted using the outgroup Rhexocercosporioides carotae (DQ526376).

Phyllosticta multicorniculata
Bissett & Palm was identified to be the only organism associated with needle-blight symptoms in A. balsamea (Fig. 1A), occurred during 1999 and 2000 in southern Québec, Canada.This is the first report of this fungal species in the province of Québec.The fungus was isolated on PDA from both healthy and symptomatic needles.From a total of 160 symptomatic needles per year, 62% were colonized in 1999 and 68% in 2000.description: All morphological characters of this fungal specimen agrees with B i s s e t t and P a l m 's (1989) concept of Phylossticta and the P. mul ticorniculata description, in having immersed unilocular and ostiolate pycnidia (Fig. 1B, 1C), and unicellular and multi appendaged mucilaginous conidia (Fig. 1D).In this study, two kinds of conidiogenous cells were found in conidiomata cavity: i) those producing macroconidia, lining the base and most of the side walls and, ii) those producing microconiogenous cells, lining the ostiolar channel (Fig. 2A).It is important to emphasize that the occurrence of microconidiogenesis in P. multicorniculata is an unusual feature for this  1974) and N a g R a j (1993) should be consulted.culture: on PDA, the fungus produced an olivaceus to greenish black culture with white margin (Fig. 4A).Colonies were of slow growth (approx.3 cm diam.for 2 weeks at 21°C in darkness) forming dense mycela.Hyphae were hyaline or slightly coloured, 2-4 μm in diameter, septate and usually forked at the ends (Fig. 4B).Pathology: In vitro, all three tested P. multicorniculata isolates showed pathogenic properties on balsam fir causing tissue necrosis of artificially infected seeds and seedlings (Fig. 4C).

diScUSSion
In Canada, P. multicorniculatis is currently known to exist with limited distribution in ontario (loc.Muskoka) and Prince edward Island (loc.Qeens), occurring exclusively on the Abies balsamea (Fig. 5).The present study clearly indicated that the fungus also occurred on balsam fir in southern Québec (loc.Sherbrooke), 50 km near the Maine, US.
The fungus has been reported previously in US, but only on White fir (Abies con color) in the region of Wisconsin (We n n e r, 1997).The real distribution, origin, ecology as well as impact of this fungus on forest ecosystems in Canada are still unknown.This is unfortunate considering a proved pathogenic behaviour of the fungus on seed of balsam fir in vitro as well as mature trees in natural forests.
Phylogenetic position of P. multicorniculatis is also still unclear.This preliminary study, based on analyses of ITS rDNA region, shoved that the fungus formed distinct clade from P. abietis.In addition, the occurrence of microconidiogenesis in P. multicorniculatis is also identified.This represents an unusual feature for genus Phyllosticta, which needs further taxonomical clarification.
Although, the morphological differences between P. multicorniculatis and P. abietis are obvious, it is unknown whether these two pathogens could attack and share the same host in a specific geographical region.For example, Phyllost icta abietis Bissett & Palm was found on Grand fir (Abies grandis) in Idaho (USA) and is considered to be destructive pathogen for Christmas tree plantations (F u n k , 1985, We n n e r, 1997).
Further taxonomical and ecological studies will be therefore needed in order to fill the gap of the knowledge on Phyllosticta associated with various Abies species in North America.For that purposes, it seems appropriate to conduct a more extensive collection of fungal isolates from Abies hosts throughout Canada in order to differentiate Phyllosticta species and enlighten their taxonomy, ecology and mycogeography.This data could be use than as useful tools to develop efficient control strategy against these phytopathogenic treats for Canadian boreal forests.

conclUSion
The study is being published in order to assist diagnostics of disease problems on balsam fir trees and to provide new information for current forest protection efforts in Canadian boreal region.It also supports monitoring, an important asset against exotic pathogens within an international trade of coniferous trees throughout world.Basé sur des analyses préliminaires de la région de l'ITS, le champignon est proche des taxons similaires, qui sont distribués aux trois regroupements: Phyllosticta (les anamorphes) et Guignardia and Botryosphaeria (les téléomorphes).Cependant, la position phylogénétique de P. multicornicu lata reste non résolue, ce qui demande l'accomplissement d'une étude moléculaire plus poussée.D'ailleurs, c'est la première mention de la présence des microconidies chez P. multicorniculata et la première illustration du développement des appendices gélatineuses du macroconidie.Les données présentées représentent une contribution dans le domaine de la diagnostique de la maladie ainsi que de nouvelles connaissances sur la distribution de la maladie au Canada.