3.3.72
IDENTIFICATION OF MON1LINIA (BROWN ROT) SPECIES

L CORAZZAl, RTA COOK2, CR LANE2, CE FULTON3, GCM VAN LEEUWEN4, AM PEREIRA 5, A NAZARE-PEREIRA5, P MELGAREJO6 and A DE CAL6

lISPV, 00156 Roma, ltaly; 2 CSL, York, Y04 ILZ UK; 3Queens University, Belfast, BT9 5PX UK; 4PD, 6700HC Wageningen, NL; 5UTAD, 5000 Vila Real, Portugal; 6MA, 28040 Madrid, Spain

Background and objectives
Of the three species of Monilinia causing brown rots of top fruit, M. fructicola, causes most damage to stone fruit. Present in the Americas and Australasia but not in Europe, it is an EU listed quarantine fungus. Its morphology and biology are very similar to the two European species, M. fructigena and M. laxa. A reliable method of distinguishing these species must precede development of a rapid diagnostic technique, the objective of an EU project. Here we report a means of identification based on morphology, cross referenced to associated PCR and aerological tests.

Materials and methods
At least 30 isolates per species were collected from Europe, Australia, New Zealand, Japan and USA. Six representative isolates of each species grown on three replicate plates of potato dextrose agar at 22oC for 5 d either in the dark (D) or in 12 h light/ 12 h dark (L/D) were examined. The following critical characters were assessed: growth rate (increase in radius between days 3 and 5, recorded as fast if >10 mm), abundance, colour and presence of diurnal rings of sporulation, colony habit, and, after 6 d on oatmeal agar, strong interaction lines [3 ] between colonies of M. fructigena and other isolates. Germ tubes of conidia streaked onto tap water agar and incubated in D for 24 h were considered to be 'long' if they measured > 100 Ám to the first branch. Monilinia DNA was amplified using universal primers, ITS 40 and ITS 4 and sequenced on an automated sequencer.

Results and conclusions
M. laxa had poor to moderate sporulation lacking both diurnal rings and yellow/honey tints in L/D (very poor sporulation in D). Growth was variable usually with a 'rosetting' habit, including dark-edged lobing of the mycelium, and a strong interaction with M. fructigena. Germ tubes were usually short. M. fructigena had poor to moderate sporulation in L/D. It was often in raised concentric rings and usually tinted yellow/honey even in D. Rosetting was usually absent. Growth rate and germ tube length were variable. M. fructicola had abundant sporulation in both L/D and D. Usually in diurnal rings in L/D, it was never tinted yellow or honey in D. Growth was fast without rosetting but with a strong interaction with M. fructigena. Germ tubes were usually long. Nucleotide sequence data of ITSI and 2 unambiguously separated the isolates into three lineages conforming to the three morphologically defined species. A species specific primer designed from the 18S gene rapidly differentiated M. fructicola from the other two species [1]. The development of a rapid aerological test using monoclonal antibodies has also started [2].

Funding from MAFF UK Plant Health Division and from the Commission of the EC Agriculture and Fisheries programme (FAIR, CT95-0725) and assistance from many others in the programme are gratefully acknowledged.

References
1. Fulton CE, Brown AE, 1997. FEMS Microbiology Letters 157, 307-312.
2. Hughes KJD, et al, 1998. Abstract in ICPP98.
3. Sonoda RM, Ogawa JM, Manji BT, 1982. Plant Disease 66, 325-326.