Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas.

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.

First Report of an Aster Yellows Phytoplasma Associated with Cabbage in Southern Texas.

In early spring 2000 carrot crops in southwestern Texas were severely infected by an outbreak of phyllody associated with aster yellows phytoplasma. Cabbage crops that had been planted adjacent to these carrot fields began to display previously unobserved symptoms characteristic of phytoplasma infection. Symptoms included purple discoloration in leaf veins and at the outer edges of leaves on cabbage heads. Proliferation of sprouts also occurred at the base of the stem and between leaf layers of some plants, and sprouts sometimes continued to proliferate on extended stems. About 5% of cabbage plants in the field exhibited these symptoms. Two symptomless and four symptomatic cabbage heads were collected in early April from one cabbage field. Veinal tissues were stripped from each sample and used for total nucleic acid extraction. To obtain specific and sufficient amount of PCR products for analysis, nested PCR was performed by using primer pairs (first with P1/P7 followed by R16F2n/R16R2) (1,2) universal for phytoplasma detection. A specific 16S rDNA fragment (about 1.2 kb) was strongly amplified from the four symptomatic but not from the two asymptomatic samples. The nested PCR products obtained from the four symptomatic samples were then analyzed by restriction fragment length polymorphism (RFLP) using the restriction enzymes MseI, HhaI, and HpaII, and the RFLP patterns were compared to the published patterns of known phytoplasmas (1). The resulting RFLP patterns were identical to those of a phytoplasma belonging to subgroup B of the aster yellows phytoplasma group (16SrI). These RFLP patterns were also evident in putative restriction sites observed in a 1.5 kbp nucleotide sequence of the 16S rDNA. This is the first report of aster yellows phytoplasma associated disease symptoms in cabbage in Texas. The occurrence of cabbage proliferation coincided with the presence of high populations of the insect vector, aster leafhopper. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I. Academic Press, San Diego, CA.

First Report of a Member of Aster Yellows Phytoplasma Group and of Clover Proliferation Phytoplasma Group Associated with Onion in Texas.

An unknown disease(s) emerged this spring (2000) in an onion field in southwestern Texas. Infected onion plants exhibited two symptom types, one with shoot proliferation, moderate stunting of plants, and light yellowish discoloration on leaves (A) and the other with only severe stunting of the plants (B). The bulbs of the infected plants collected from both symptom types were smaller than normal. When the aerial shoots were trimmed, the infected (but not asymptomatic or the severely stunted) bulbs produced multiple slender sprouts after storage in room temperature for about a month. These symptoms are characteristic of yellows diseases caused by phytoplasmas. Ten symptomatic (six with symptom type A and four with symptom type B) and ten symptomless onion plants were collected in early May from an onion field about 1 to 2 weeks prior to blooming. Total nucleic acid was extracted from 0.5 g of shoot tissues from each sample. Nested polymerase chain reaction (PCR) using universal primer pairs (P1/P7 followed by R16F2n/R16R2) previously designed based on 16S and 23S rRNA gene sequence (1,2) was employed for the detection of phytoplasma(s) present in the samples. Specific PCR products (all were about 1.2 kb) were heavily amplified from five samples with symptom type A and one with symptom type B. Three of the symptomatic plants showing symptom type B and five of the symptomless samples were scored as weak positives. Restriction fragment length polymorphism (RFLP) analyses of the PCR products obtained from all five symptomatic samples with symptom type A using restriction enzymes including MseI, HhaI, and HpaII revealed that the associated phytoplasmas detected belonged to aster yellows phytoplasma group (16SrI), subgroup A. RFLP analyses of PCR product from the sample with symptom type B indicated that the associated phytoplasma belonged to clover proliferation group (16SrVI), subgroup A (1). Since symptom type A resembles onion yellows reported elsewhere, we propose to adopt "onion yellows" to refer to the new onion disease occurring in Texas. However, correlation between a member of clover proliferation phytoplasma group and onion plants showing severe stunting could not be firmly established. A phytoplasma belonging to 16SrI-B is associated with onion yellows disease reported in Japan (1). This is the first report that onion yellows occurs in the United States. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) B. Schneider et al. 1995. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I, Academic Press, San Diego, CA.

Intramammary response to modified intramammary devices.

Twenty-five cows in three experiments were used to evaluate the following intramammary devices: abraded surfaces; abraded and sulfur hexafluoride-coated; abraded, sulfur hexafluoride-coated, and weighted; and smooth surfaces and weighted. The objectives of the experiments were 1) to determine whether coating the abraded intramammary devices prevented amorphous deposits and bacterial adherence on the devices, 2) to determine whether addition of weight to devices increased the concentration of somatic cells in milk, and 3) to evaluate milk production and response of mammary epithelial cells to the devices. Milk SCC and NAGase (EC 3.2.1.30) from quarters fitted with devices were increased in quarter bucket and stripping milk, but increases were similar among all devices. Macrophages were the predominate cell type in quarter milk before insertion and neutrophils after insertion. Moderate to heavy deposits of amorphous material were observed on all devices, regardless of time residing in the gland. Approximately 50% of the devices were colonized after intramammary inoculation with Corynebacterium bovis. Milk production in control quarters and in quarters with devices were similar. Neither coating with sulfur hexafluoride nor addition of weight to devices reduced amorphous deposits or bacterial adherence or enhanced milk somatic cell response.