During this study, we focused on 1) improving and validating a rapid assay for detection of AY MLO based on DNA hybridization (the polymerase chain reaction technique, PCR) and 2) gathering preliminary data on susceptibility of lettuce varieties to AY MLO in the field.

Two sets of DNA primers (oligonucleotides of known sequence) designed for general detection of MLOs (primers r16F2/r16R2) or for specific detection of AY MLO (primers r16F4/r16R1) were tested. The primers were obtained from R. E. Davis, USDA-ARS, Beltsville, MD. The general primer proved to give inconsistent results in the assay, so all results reported are based on use of the AY MLO-specific primer r16F4/r16R1. A rapid DNA extraction method was developed, allowing the preparation of insect samples for analysis in a few minutes. We were also able to improve the sensitivity of the assay at least 8-fold by including a second polymerase enzyme. The assays could be completed in one day, although typically the PCR reaction was run overnight, and products were detected the next day. This represents a significant improvement over the bioassay.

The PCR assay was tested and compared with the bioassay using insects collected in commercial lettuce fields in Celeryville and Hartville, Ohio biweekly throughout the summer, for a total of six collections (Table 1). Whenever possible (when populations permitted), at least 100 leafhoppers were collected from each location, then brought to the laboratory in Wooster where they were placed on individual lettuce seedlings in small cages. After an inoculation access period of 7 days, leafhoppers were removed from their cages, bulked in groups of 8-10, extracted and tested by PCR. The proportion of positive individuals in the bulked samples was estimated using the formula: z = 1 - (1-x)1/y , where z = the proportion of positive insects, x = the proportion of positive groups of insects and y = the number of groups of insects. Lettuce plants were held for an additional 2 wk, then scored for the presence of AY symptoms. Results shown in Table 1 indicate that the PCR assay usually resulted in higher proportions of insects infected by AY MLO than the bioassay. Our previous studies have shown that AY MLO can be detected in leafhoppers soon after exposure, before they have completed the latent period and become inoculative. These data may reflect the detection of the MLO in infected but non-inoculative leafhoppers by the PCR technique. Bioassay values may also be lower than expected due to disease escapes. For collections made on July 22 and Aug 8 in Celeryville, all of the bulked insect samples tested by PCR were positive, resulting in an estimated proportion of MLO-positive individuals of 100%. Since our previous studies have shown that only one AY MLO-infected leafhopper in a sample of 10 can be detected by PCR, this estimation is very likely to be too high. We will address this issue in future validation studies by using more samples consisting of fewer insects.

Table 1. Detection of aster yellows (AY MLO) in leafhoppers (Macrosteles quadrilineatus) collected in Celeryville and Hartville, Ohio, in 1994 by bioassay and the PCR technique.

The relative susceptibility of lettuce and escarole varieties to AY was evaluated in a field trial carried out in Celeryville, OH at the Muck Crops Branch of OSU-OARDC. Lettuce seedlings were raised in the greenhouse at the OARDC facility in Wooster, then exposed to 120 AY MLO-inoculative leafhoppers (bolt or severe strain) per flat of 96 plants. Seventeen varieties of lettuce and one of escarole were tested in a randomized complete block design, with 25 plants per replicate, three replicates per treatment. Plants were rated for disease incidence 31 and 41 days after transplanting. The results of the trial are shown in Table 2. All of the lettuce varieties tested were susceptible to aster yellows, while the escarole was highly resistant or immune. Some of the plants had been exposed to only 50 leafhoppers/flat, and these had much less disease than plants exposed to 120 leafhoppers/flat. Both strains of AY MLO caused high disease incidence on the lettuce varieties.

Table 2. Incidence of aster yellows 31 d after inoculation of lettuce varieties with either the "bolt" or "severe" strain of aster yellows mycoplasma-like organism.

	Mean Disease Incidence
	(Percent Diseased Plants)
Variety	Bolt Strain	Severe Strain
Vanity MTO	79.6	81.6
Butterhead Boston	18.2*	81.1
Red Butterhead	36.3*	93.8
Red Salad Bowl	81.2	74.9
Fancy Butterhead Boston	89.7	86.4
Tall Guzmaine	85.4	92.5
Esmerelda	39.9*	82.3
Special Ideal Cos Mi	62.2	57.6*
Slobolt	57.8	72.0
New Fire Red	70.1	82.9
Pic 318 Romaine	69.9	81.2
New Red Fire	68.0	90.2
Summer Bib Mi	72.3	23.4*
Esmerelda Mi	92.1	88.1
Slo Bolt Mi	42.4	64.0
Waldman's Green Leaf	93.6	82.5
Valmaine	86.7	87.4
Deep Heart Escarole	0.0	0.0*
*Indicates transplants inoculated with 30-50 leafhoppers/flat; 120 leafhoppers/flat used for all other inoculations.

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