1Department of Phytopathology, Wageningen Agricultural University, P.O. Box 8025, 6700 EE Wageningen, the Netherlands; 2Plant Protection Service, P.O. Box 9102, 6700 HC Wageningen, the Netherlands

Background and objectives
Worldwide, the brown rot fungi cause considerable losses in pome and stone fruit growing areas. Three species are being distinguished within the brown rot fungi, Monilinia fructigena, M. laxa and M. fructicola (latter is a quarantine organism for Europe). Species separation is mainly based on growth/colony characteristics on agar media [1]. However, due to considerable variation in natural media, the species- specific colony characteristics described in literature are not always encountered [2]. Furthermore, diagnostic personnel not familiar with Monilinia cultures, find difficulties in identification even with more or less typical cultures. The typical characteristics for the three species are mainly qualitative traits (e.g. shape of the colony margin, colour of sporogenous tissue), which can be ambiguously interpreted. It was our aim to define and assess several quantitative colony and germ tube characteristics for the three brown rot fungi.

Materials and methods
Isolates obtained from different regions (Europe, USA, Australia, Japan) were identified on basis of general (qualitative) colony characteristics on PDA (Oxoid). Growth rate and sporulation intensity was studied on 4 % PDA (Oxoid) in a light/dark regime (nUV light, Philips TLD 18W/08) at 22C with three replicate Petri dishes (9 cm) per isolate. Colony diameter was measured after 3 and 5 days of incubation, sporulation intensity was determined by washing the plates after 14 days of incubation and determining the number of conidia with a haemocytometer. For the study of germ tube morphology, conidia were obtained from 4-14 days old PDA plates, incubated under nUV light at 22C. Water agar plates (1.5 %) were seeded with 0.1 ml of a conidial suspension adjusted to 1x105 spores/ml. Incubation took place for 18 h in darkness at 22C, after which measurements were done at 100x magnification (50 conidia per isolate). Three germ tube characteristics were determined : the distance from conidium to the first branch in the germ tube (branch at least 20 m long), the distance from conidium to the tip of the germ tube (longest straight length), and the absencelpresence of more than one germ tube per conidium. A discriminant analysis was performed on the basis of several combinations of the five quantitative characteristics, and the number of misclassifications was determined.

Results and conclusions
In all M. fructicola isolates except one, increase of colony diameter from day 3 to 5 was higher than this for M. laxa and M. fructigena. The slowest growing M. fructicola isolate showed overlap with some of the M. fructigena isolates. Sporulation intensity was by far highest in M. fructicola (range 8.5x106 to 38.4x106 conidia per Petri dish). Mean length of the germ tube after 18 h was consistently higher for M. fructicola compared with M. laxa. In M. fructicola mean length ranged from 465 to 851 m, in M. laxa this was 161-466 m. A typical characteristic for M. fructigena was the high percentage of conidia with more than one germ tube (ranging from 14 to 74%). Discriminant analysis showed that classification on basis of colony growth rate and mean length of the germtube gave good results; only 2 misclassifications occurred out of 29 isolates tested. When sporulation intensity was added in the analysis, only one misclassification occurred. We conclude that some simple, unambiguously defined quantitative characters can be of great help in diagnostics to delineate the three brown rot fungi of fruit crops.

1. Byrde RJW, Willetts HJ, 1977. The brown rot fungi of fruit. Their biology and control. Pergamon Press, Oxford, 171 pp.
2. Ogawa JM, Gilpatrick JD, Uyemoto JK, Abawi GS, 1978. Plant Disease Reptr. 62, 437-441.