In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA Ile and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.

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The phylogenetic relationships of 17 phytopathogenic mycoplasmalike organisms (MLOs) representing seven major taxonomic groups established on the basis of MLO 16s ribosomal DNA (rDNA) restriction patterns were examined by performing a sequence analysis of the 16s rDNA gene. The sequence data showed that the MLOs which we examined are members of a relatively homogeneous group that evolved monophyletically from a common ancestor. In agreement with results obtained previously with other MLOs, our results also revealed that the organisms are more closely related to AchoZepZasma Zaidhwii and other members of the anaeroplasma clade than to any other mollicutes. A phylogenetic tree based on 16s rDNAs showed that the MLOs which we examined can be divided into the following five primary clusters: (i) the aster yellows strain cluster; (ii) the apple proliferation strain cluster; (iii) the western-X disease strain cluster; (iv) the sugarcane white leaf strain cluster; and (v) the elm yellows strain cluster. The aster yellows, western-X disease, and elm yellows strain clusters were divided into two subgroups each. MLOs whose 16s rDNA sequences have been determined previously by other workers can be placed in one of the five groups. In addition to the overall division based on 16s rDNA sequence homology data, the primary clusters and subgroups could be further defined by a number of positions in the 16s rDNAs that exhibited characteristic compositions, especially in the variable regions of the gene.Nonhelical phytopathogenic mollicutes, which are most often referred to as mycoplasmalike organisms (MLOs), are wall-less, nonculturable prokaryotes that cause diseases in several hundred plant species (23). The first attempts to differentiate and classify these organisms were based on symptoms, host ranges, and insect vector relationships (4, 9, 21).The phylogenetic interrelatedness of the MLOs was poorly understood until recently, when molecular methods were introduced into plant mycoplasmology. Many MLO strains were differentiated and partially characterized by dot and Southern hybridization techniques, as well as by serological techniques. Most of this work has been reviewed by Kirkpatrick (10) and Lee and Davis (16). Closely related MLOs belonging to the aster yellows strain cluster could be classified by Southern blot analysis (17).Neither serological methods nor nucleic acid hybridization experiments performed with randomly cloned DNA fragments have revealed the phylogenetic or taxonomic positions of MLOs in relation to each other and to other microorganisms. In contrast, the 16s rRNA gene is a universal characteristic in prokaryotes and has both conserved and sufficiently variable regions. This gene is therefore suitable for phylogenetic and taxonomic classifications at various levels, including intrageneric differentiation (30,34 yellows agent (strain SAY), and the western-X disease agent (strain WX), as well as several Japanese MLOs, showed that these organisms exhibit levels of sequence homology of at least 89.4%. Thus...

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Bioassays were used to demonstrate the antibiotic effect of Trichoderma isolates on P. cactorum. When both fungi were grown on benomyl‐containing PDA medium, the mycelial growth of Trichoderma was suppressed. However, the production of antibiotics by this fungus remained active, leading to inhibition of the mycelial growth of P. cactorum. The antibiotic effect of Trichoderma on zoospores and cysts was tested on a PDA substrate precultured with Trichoderma on cellophane sheets. On the substrate of some Trichoderma isolates, lysis of zoospores, formation of extracellular vesicles, and hypertrophy of the water expulsion vesicle did occur, both resulting in the death of the zoospores. Conidial suspensions of Trichoderma isolates also induced zoospore lysis. It is presumed that membrane‐active peptide antibiotics (peptaibols) are involved in zoospore lysis. The peptaibol paracelsin caused lysins of zoospores at a concentration of 2.5 × 10−4 M. The effect on cysts depended on the Trichoderma isolate tested and the age of Trichoderma preculture. Old cultures (after beginning of sporulation) affected cysts more severely than young cultures (before sporulation) which usually were not lethal to the cysts but induced preferably microsporangium formation, inhibition of cyst germination, and retardation of germ tube growth.

Mycoplasma‐like organisms (MLOs) were constantly detected by the DAPI technique and by restriction fragment length polymorphism (RFLP) analysis of PCR‐amplified DNA in trees of Pyrus pyrifolia cvs Hosui and Kosui grafted on P. communis, seedlings rootstock with symptoms similar to the slow form of pear decline. These symptoms included upward curling of the leaves along the midrib. Leaves were abnormally thick and later turned reddish while major veins became swollen and brown. Trees with symptoms were usually 4–5 years old and were growing in the major pear areas of central Italy. The incidence of affected trees was particularly high in one orchard adjacent to a pear orchard strongly affected with the slow form of pear decline. In this case the distribution pattern of affected Nashi trees suggests that the causal agent was introduced from the adjacent pear orchard by an aerial vector. Although oriental pears are well‐known hosts of the pear‐decline agent when used as rootstocks of French cultivars, this is the first report of pear decline in P. pyrifolia varieties.