Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.     

<Emphasis Type="Italic">In vitro</Emphasis> screening for sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>L.) resistant calli to <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> fungal culture filtrate

Diaporthe helianthi the causal agent of sunflower (Helianthus annuus) stem canker, causes significant reductions in yield and oil content in most sunflower-growing areas. With the aim of enhancing host resistance, we selected in vitro sunflower calli against culture filtrates of two pathogen isolates (7/96 and 101/96). This technique may be an effective and rapid tool to discriminate the most virulent D. helianthi isolate and to screen for host resistance in the early stage of a breeding programme. Further investigation on the mechanisms involved in defence pathways showed no induction of salicylic acid and pathogenesis-related proteins in calli, indicating that the host resistance is not associated with Systemic Acquired Resistance but probably other biochemical mechanisms.     

Root and stem rot of chrysanthemum caused by five <Emphasis Type="Italic">Pythium</Emphasis> species in Japan

Root and stem rot with wilt of above ground parts of cultivated chrysanthemums was first found in Ibaraki, Toyama and Kagawa prefectures, Japan in 2002 and 2003. Pythium species were isolated from the diseased tissues and identified as P. dissotocum, P. oedochilum, P. sylvaticum, P. ultimum var. ultimum and asexual strains of P. helicoides based on their morphologies and sequences of rDNA-ITS region. All the Pythium species were strongly pathogenic to chrysanthemums in pot conditions and were reisolated from the inoculated plants. Because Pythium root and stem rot of chrysanthemum has never been reported in Japan, we propose that this is a new disease that can be caused by the five Pythium species.     

Effect of indole-acetic acid (IAA) on the development of symptoms caused by <Emphasis Type="Italic">Pythium ultimum</Emphasis> on tomato plants

The effect of indole-acetic acid (IAA) on the development of symptoms caused by Pythium ultimum on tomato plants was investigated using different bioassays. Application of IAA (5 μg ml⁻¹) on tomato seedlings inoculated with P. ultimum did not affect their emergence suggesting that IAA did not affect the severity of Pythium damping-off. However, IAA was shown to influence the development of P. ultimum symptoms on tomato plantlets. Low concentrations of IAA (0–0.1 μg ml⁻¹) within the rhizosphere of plantlets increased the severity of the symptoms caused by P. ultimum, while higher concentrations (10 μg ml⁻¹), applied either by drenching to the growing medium or by spraying on the shoot, reduced the symptoms caused by this pathogen. In addition, the study demonstrated that P. ultimum produces IAA in liquid culture amended with L-tryptophan, tryptamine or tryptophol (200 μg ml⁻¹) and in unamended culture.     

Isolation and characterization of lectins from the AG-D group of binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> species

Isolates from 18 anastomosis groups (AGs) of binucleate Rhizoctonia were screened for lectin activity. Eight AGs (AG-B, AG-D, AG-F, AG-G, AG-H, AG-I, AG-R and AG-U) had low to moderate lectin activities. Among these, members of AG-D and AG-I had the highest activity. Partially purified lectins from AG-D preferentially agglutinated human blood type A to type B and O. Mucin and galactose were the most potent inhibitors among the tested carbohydrates. The molecular masses of these lectins ranged from 12.7 kDa for the monomer to 62 kDa for the pentamer type. Proline, alanine, glutamic acid, aspartic acid, leucine, threonine, serine and tyrosine were the major amino acid components of these lectins. Lectins from AG-D were stable at 4–50°C and from pH 6.0 to 10.0. When assayed with isoelectric focusing, these lectins gave bands at pI 9.30. Specificity of lectins from AG-D to galactose and its derivatives suggest a possible recognition role in this fungal species.     

Ineffectiveness of pruning to control citrus huanglongbing caused by <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter americanus

The huanglongbing (HLB) disease of citrus trees, caused by Candidatus Liberibacter asiaticus and Ca. Liberibacter americanus, was first reported in Brazil in March, 2004. The presence of the disease has caused serious concerns among growers. Pruning experiments were conducted to determine if removal of symptomatic branches or the entire canopy (decapitation) would eliminate infected tissues and save HLB-affected trees. Pruning was done in five blocks on a total of 592 3- to 16 year-old ‘Valência’, ‘Hamlin’ or ‘Pêra’ sweet orange trees showing no symptoms or with two levels of symptom severity. Ten decapitated trees per block were caged and all trees were treated with insecticides to control the psyllid vector, Diaphorina citri. Mottled leaves reappeared on most symptomatic (69.2%) as well on some asymptomatic (7.6%) pruned trees, regardless of age, variety, and pruning procedure. Presence of the pathogen (Ca. Liberibacter americanus) in all symptomatic trees was confirmed by PCR. In general, the greater the symptom severity before pruning the lower the percentage of trees that remained asymptomatic after pruning.     

Molecular characterization of <Emphasis Type="Italic">Phoma tracheiphila</Emphasis>, causal agent of Mal secco disease of citrus,in Israel

Mal secco disease of citrus caused by Phoma tracheiphila is a devastating disease in the Mediterranean basin. Susceptible citrus species include lemon, citron, lime and others. Trees attacked by the fungus show characteristic symptoms; the smallest twigs die first, followed by the larger branches. Eventually, the whole tree is killed. The symptoms are clear in the orchards but by the time they are visible the disease is already well established. The need for a sensitive, reliable and rapid diagnostic method for the early identification of the fungus in trees and fruit exists. We have developed a PCR-based method for the identification of P. tracheiphila from plant tissues including fruit. Any such method must take into account the genetic variability in the pathogen population. Molecular methods were used to compare different isolates of P. tracheiphila. This study found no significant differences between different isolates from different citrus species from different parts of Israel.     

Cloning of DNA fragments specific to the pathotype and race of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Japanese isolates of Verticillium dahliae, a causal agent of wilt disease in many plants, are classifiable into pathotypes based on their pathogenicity. Because these pathotypes are morphologically indistinguishable, establishing a rapid identification method is very important for the control of this pathogen in Japan. For cloning DNA fragments that are useful for identification and specific detection of V. dahliae pathotypes, we performed random amplified polymorphic DNA (RAPD) analyses using various isolates. One polymerase chain reaction (PCR) product, E10-U48, was specific to isolates pathogenic to sweet pepper. The other product, B68-TV, was specific to race 1 of isolates pathogenic to tomato. The specificity of these sequences was confirmed by genomic Southern hybridization. Further analyses revealed that the region peripheral to B68-TV obtained from the genomic DNA library includes the sequence specific to all isolates pathogenic to tomato (races 1 and 2). Moreover, sequence tagged site (STS) primers designed from B68-TV and its peripheral region showed race-specific and pathotype-specific amplification in a PCR assay. The probes and primers obtained in this study are likely to be useful tools for the identification and specific detection of pathotypes and races of V. dahliae. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession number AB095266.     

PCB removal,soil enzyme activities,and microbial community structures during the phytoremediation by alfalfa in field soils

Purpose  

An in situ phytoremediation trial was developed in order to investigate the function of alfalfa during a 2-year bioremediation of an agricultural soil contaminated with polychlorinated biphenyls (PCBs). The study was conducted with the aim to better understand the application potential of PCB phytoremediation at field scale.