SUMMARY Quantitative expression of Sclerotinia sclerotiorum genes encoding two endo-polygalacturonase (endo-PG) isoforms (PGa and PGb), malate dehydrogenase (MDH, a key enzyme in fungal biosynthetic pathway of oxalic acid), and plant polygalacturonase-inhibiting protein (PGIP) were monitored by real-time reverse transcrip- tion-polymerase chain reaction (qRT-PCR) during the early stages (0-48 h) of soybean seedling infection. The activity of the two endo-PGs was also investigated dur- ing plant infection. PGa and PGb activity reflected very closely the pattern of their transcript accumulation as determined by qRT-PCR. In particular, the PGb encod- ing gene (Sspgb) was induced at 8 h after inoculation and reached a maximum at 16 h; expression of the PGa encoding gene (Sspga) was comparatively lower, reach- ing its maximum level later and its rate of increase par- alleled that of the S. sclerotiorum b-tubulin gene; the ex- pression of the MDH encoding gene (Ssmdh) was maxi- mal 16 h after infection; soybean pgip transcript began to accumulate 8 h after inoculation reaching a maxi- mum after 24 h. Expression patterns of reported genes are discussed in relation to the ability of S. sclerotiorum to induce disease by regulating endo-PGs and oxalate accumulation to elude the effect of plant PGIP. Key words: endopolygalacturonase, PGIP, soybean, oxalic acid, real-time RT-PCR. INTRODUCTION Sclerotinia sclerotiorum is a necrotrophic pathogen able to infect a wide range of host plants (Boland and Hall, 1994). During pathogenesis, this fungus secretes a large number of cell-wall degrading enzymes (CWDEs) to penetrate, colonize and macerate host tissues (Mar- ciano et al., 1983; Riou et al., 1991). Among CWDEs, Corresponding author: F. Favaron Fax: +39.49.8272890 E-mail: francesco.favaron@unipd.it endo-polygalacturonase (endo-PG) produced by S. sclero- tiorum is able to macerate intact tissue (Favaron et al., 1993). The endo-PG of S. sclerotiorum is encoded by sev- eral pg genes which, based on sequence similarity, can be classified into four groups. The first group encodes neu- tral or basic isoforms and comprises three genes (sspg1a, sspg1b, sspg1c) isolated from strain S5 of S. sclerotiorum (Reymond et al., 1994; Fraissinet-Tachet et al., 1995) and two additional ones, sspg1d and Sspgb, isolated from strains 100 and B-24, respectively (Favaron et al., 2004; Li et al., 2004b). The second group encodes acidic PG iso- forms and includes three genes (pg5, sspg5 and Sspga iso- lated from S5, 100 and B-24 strains, respectively) showing a high degree of sequence similarity (Favaron et al., 2004; Kasza et al., 2004; Li et al., 2004b). The third and fourth group code for acidic PGs with a peculiar N-terminal se- quence and comprise the pair pg6/sspg6 and the pair pg7/sspg3, respectively. The genes pg6 and pg7 were isolat- ed from strain S5 while the genes sspg3 and sspg6 were isolated from strain 100 (Kasza et al., 2004; Li et al., 2004b). Only the Sspga, Sspgb and sspg1b gene products have been characterized (Cotton et al., 2002; Favaron et al., 2004) and analysis of gene expression performed by RT-PCR showed that the basic or neutral PGs are the first to be expressed by S. sclerotiorum when infecting host plants (Favaron et al., 2004; Kasza et al., 2004). Previously, we have found that some biochemical properties of PGb, encoded by Sspgb, support its prominent role at an early stage of soybean seedling in- fection (Favaron and Marciano, 1992; Favaron et al., 1992, 1993). PGb also induces programmed cell death on soybean cells, an important property for a necro- trophic fungus such as S. sclerotiorum (Zuppini et al., 2005). In addition, we observed that oxalic acid secret- ed by S. sclerotiorum during plant infection has a dual effect on PGb activity. Initially, it changes the pH to- ward more suitable values for PG activity and then, at sub-optimal pH, has a direct enhancing effect on the ac- tivity of the enzyme (Favaron et al., 2004). We observed also that PGa, the acidic isoform encoded by the Sspga gene, escapes inhibition by soybean polygalacturonase- inhibiting protein (PGIP) at acidic pH determined at later stages of soybean infection. Based on these observations, we hypothesized that S. Journal of Plant Pathology (2005), 87 (3), 199-205 Edizioni ETS Pisa, 2005 199 EXPRESSION OF TWO SCLEROTINIA SCLEROTIORUM ENDO-PG GENES CORRELATES WITH ENDO-POLYGALACTURONASE ACTIVITY DURING GLYCINE MAX COLONIZATION L. Sella 1 , A. Tomassini 1 , R. D’Ovidio 2 and F. Favaron 1 1 Dipartimento del Territorio e Sistemi Agro-Forestali, Sezione di Patologia Vegetale, Università degli Studi di Padova, Viale dell’Università 16, I-35020 Legnaro, Italy 2 Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via S. Camillo de Lellis, I-01100 Viterbo, Italy