‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3-ends, but also the 5-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Detection of the Phoma pathogens Plenodomus biglobosus subclades ‘brassicae’ and ‘canadensis’ on wasabi,and ‘canadensis’ in Europe

European Journal of Plant Pathology - Phoma stem canker / blackleg is an internationally important disease of Brassicas including B. napus (oilseed rape, OSR), caused by multiple genetic subclades...     

Molecular diversity of ‘Candidatus Phytoplasma pyri’ isolates in Slovenia

A European quarantine organism ‘Candidatus Phytoplasma pyri’ causing devastating pear decline disease has been reported to affect pear trees in several European countries. In this study a multilocus sequence analysis was successfully used to gain detailed insight into the molecular diversity of thirty closely related ‘Candidatus Phytoplasma pyri’ isolates from different orchards in Slovenia. Among three genomic regions analyzed, the 16S/23S rRNA intergenic spacer region was the most conserved among Slovenian isolates with 99.7 % sequence identity, yielding only three distinct genotypes. On the other hand, five different genotypes were detected when analyzing secY and aceF genomic regions that shared sequence identity of 94.8 and 97.2 %, respectively. Six of the detected genotypes, specifically four in the secY region and one in each of the two other analyzed genomic regions, were unique for Slovenia. At least eight different haplotypes were found with multilocus sequence analysis, indicating high molecular diversity among Slovenian ‘Ca. P. pyri’ isolates. Haplotypes were clustered into two major clusters, separated by at least 45 mutations. No connection was established between haplotype occurrence and cultivar type.     

‘Candidatus Phytoplasma ziziphi’ associated with Sophora japonica witches’ broom disease in China

Chinese scholar tree (Sophora japonica) with witches’ broom symptoms was observed in Shandong Province in China. Phytoplasmas were detected in the diseased plants using 16S rDNA amplification with phytoplasma-specific universal primer pairs. On the basis of the results of 16S rDNA sequencing, virtual restriction fragment length polymorphism patterns and phylogenetic analyses, the phytoplasma found in S. japonica with witches’ broom symptoms was confirmed as a ‘Candidatus Phytoplasma ziziphi’-related strain belonging to the Elm yellows group 16SrV. This is the first report of ‘Ca. P. ziziphi’ infecting S. japonica plant with witches’ broom symptoms.     

Comment on ‘In Turkish wheat cultivars the resistance allele of LR34 is ineffective against leaf rust’

Journal of Plant Diseases and Protection -     

Seasonal progression of leaf rust in ‘Niagara Rosada’ grapevine in a biannual crop system in Brazil

The soil-borne fungus Fusarium oxysporum can cause both Fusarium yellows and Fusarium root rot diseases with severe yield losses in cultivated sugar beet. These two diseases cause similar foliar symptoms but different root response and have been proposed to be caused by two distinct F. oxysporum formae speciales. Fusarium yellows, caused by F. oxysporum f. sp. betae, presents vascular discoloration, whereas Fusarium root rot, due to F. oxysporum f. sp. radicis-betae, appears as black rot visible on the root surface. The aim of this work was to study the host-pathogen interaction between sugar beet lines and isolates originally characterized as Fusarium oxysporum f. sp. betae. Eight susceptible sugar beet lines, selected by the USDA-ARS (US) and UNIPD (University of Padova, Italy) breeding programs, were inoculated with three different isolates of F. oxysporum f. sp. betae, the causal agent of Fusarium yellows, representing different genetic groups. All inoculated lines developed symptoms, but severity, expressed as area under the disease progress curve (AUDPC), differed significantly (P < 0.05) among lines. Two lines from UNIPD, 6 and 9, were the most susceptible to the disease, whereas the other lines showed similar levels. The three isolates of F. oxysporum f. sp. betae differed significantly (P < 0.05) in disease severity. Five weeks after inoculation the plants were harvested and roots examined. Surprisingly, severe root rot was observed in the susceptible UNIPD lines when inoculated with all three isolates, while this symptom was never observed in the USDA germplasm. The development of this disease symptom obviously depends on the plant genotype.     

Correction to: Haplotypes of ‘Candidatus Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

European Journal of Plant Pathology -     

Hexanoic acid provides long-lasting protection in ‘Fortune’ mandarin against Alternaria alternata

Alternaria brown spot disease is a serious disease in mandarins and their hybrids without effective disease control measures. In recent years, induced plant resistance has been studied as an alternative to classical pesticides, but few studies on the effectiveness of these products and their long-lasting effects in woody crops have been performed. After two inoculations with Alternaria alternata, citrus plants that were treated with hexanoic acid showed enhanced resistance, displaying lower levels of disease incidence associated with an activation of the jasmonic acid pathway, the accumulation of phenolic compounds and the expression of defensive genes, such as polygalacturonase-inhibiting proteins.     

‘Candidatus Liberibacter solanacearum’ detected in Trioza urticae using suction trap-based monitoring of psyllids in Germany

Journal of Plant Diseases and Protection - Psyllids are small, phloem-feeding insects. Several species are vectors of economically important pathogens, such as ‘Candidatus Liberibacter...     

Identifying potential novel resistance to the foliar disease ‘Scald’ (Rhynchosporium commune) in a population of Scottish Bere barley landrace (Hordeum vulgare L.)

Journal of Plant Diseases and Protection - Barley ‘Scald’ is an economically damaging fungal disease that is a global problem, causing significant yield and economical losses in the UK...     

First report of ‘Candidatus Phytoplasma asteris’ infecting hydrangea showing phyllody in Japan

Phytoplasma-induced floral malformations such as virescence, phyllody, and proliferation were observed on hydrangeas in Gunma Prefecture, Japan. Phylogenetic analyses based on 16S rRNA, secY, groEL, and amp gene sequences indicated that the affected hydrangea plants were associated with phytoplasmas belonging to ‘Candidatus Phytoplasma asteris’, but not to ‘Ca. P. japonicum’, which occurs in hydrangeas showing phyllody in Japan. This is the first molecular evidence of an association of ‘Ca. P. asteris’ with hydrangea plants in Japan.     

Molecular analysis of ‘Candidatus Phytoplasma aurantifolia’ associated with phytoplasma diseases in Myanmar

During 2010 and 2011, typical phytoplasma disease symptoms such as little leaves, phyllody and witches’ brooms were observed on black gram, green gram, long bean, shaggy button weed and sesame plants from different regions of Myanmar. The symptomatic samples were analyzed by PCR using universal phytoplasma primers and characterized by sequencing 16S rRNA, ribosomal protein and translocase protein genes. Based on sequence and phylogenetic analysis of the three genes, the phytoplasmas associated with those plants belonged to members of ‘Candidatus Phytoplasma aurantifolia’. To our knowledge, black gram and shaggy button are new hosts for ‘Ca. P. aurantifolia’.     

‘Candidatus Phytoplasma aurantifolia’-related strain associated with tomato yellows disease in China

A new disease of tomato plants with typical phytoplasma disease symptoms such as stunting, yellows, auxiliary shoot proliferation and phyllody was observed in Yunnan Province, southwest China in 2011. By a nested-PCR, phytoplasma were detected using the phytoplasma universal primers specific for 16S rDNA. The results of the 16S rDNA sequencing, computer-simulated RFLP patterns and phylogenetic analysis indicated that the phytoplasma associated with the diseased tomato plants belongs to subgroup A of the peanut witches’-broom group. This is the first report of a 16SrII-A phytoplasma associated with a new tomato disease in China. This new disease was named tomato yellows.     

No detection of seed transmission of citrus tatter leaf virus in ‘Meyer’ lemon

Journal of Plant Diseases and Protection - Citrus tatter leaf virus (CTLV), a strain of apple stem grooving virus, is a virus of citrus that is of commercial importance for trifoliate orange...     

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print-based qPCR and immunoassay

‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR-based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE-qPCR) was compared to DNA extraction of plant tissue followed by PCR (X-PCR) by testing the same tissue samples. The former estimated a higher CLas population in tissue prints than the latter (t-test; P = 0.009). All extracts prepared for TPE-qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE-qPCR and immunoassay. Although the detection rate by TPE-qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE-qPCR. Thus, a higher detection rate would be achieved by combining TPE-qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.     

Active ingredient contents of ‘me-too’ registered abamectin products and differences in their efficacy onTetranychus cinnabarinus

Many agrochemical companies in Turkey purchase generic active ingredients (a.i.) and, by way of ‘me-too’ registration, sell pesticides under different brand names. Farmers and researchers often claim that these products are ineffective. In order to determine the a.i. content and efficacy of me-too registered abamectin-based products, we studied seven (in 2004) and ten (in 2005) products, which were chosen based on their availability and high market share. Differences in both efficacy and a.i. contents were observed in the products investigated in the study, but making speculative statements that could affect all me-too registered products based on our investigations of only 17 abamectin-based samples is not realistic. Timectin® was found to be statistically less effective onTetranychus cinnabarinus in both laboratory and greenhouse trials than all other tested products. Furthermore, Timectin was determined to have 69.8% less a.i. than in its product specifications. Our findings make it clear that grower complaints about the qualities of agricultural pesticides are based on actual chemical inadequacies of formulated pesticides. Analysis of the a.i. should be carried out as part of regular market inspection in order to determine whether agricultural pesticides comply with their product specifications. In this way growers will be provided with good quality but inexpensive products.