Pollen as a possible pathway for the dissemination of Pseudomonas syringae pv. actinidiae and bacterial canker of kiwifruit

Pollen collected in a kiwifruit orchard with symptoms of bacterial canker and naturally contaminated by Pseudomonas syringae pv. actinidiae (Psa), was used to pollinate an experimental orchard, in order to confirm its role, under commercial orchard conditions, in disseminating the pathogen and, possibly, contributing to disease spread. A pollen lot, certified free from Psa, was used with the same methods as a control. Two pollination techniques were used: dusting (dry pollen) and spraying (pollen suspension in water). The orchard was monitored during 2 years from experimental pollination, with regular sampling of flowers, fruits, leaves, and vines, to check for Psa as an epiphyte or endophyte, and for bacterial canker symptoms. Psa was recovered from flowers, fruitlets and leaves during the first season, mainly in plots where contaminated pollen had been sprayed in water suspension. From early August until harvesting time (mid-October), Psa detection was possible only on leaves. No symptoms developed during the first season after pollination. No endophytic Psa was detected in pruned vines in the following winter. During the second season, detection and isolation of Psa was erratic, but direct isolation was achieved from four plots. During the second season after pollination, typical leaf symptoms were observed on a few vines, and Psa was isolated and identified. Our results suggest that Psa could be disseminated via contaminated kiwifruit pollen as a pathway for spread of bacterial canker. However, further pollination experiments are needed to establish, beyond any doubt, whether contaminated pollen may contribute to possible disease outbreaks

Pollen as a possible pathway for the dissemination of Pseudomonas syringae pv. actinide and bacterial canker of kiwifruit

Pollen collected in a kiwifruit orchard with symptoms of bacterial canker and naturally contaminated by Pseudomonas syringae pv. actinidiae (Psa), was used to pollinate an experimental orchard, in order to confirm its role, under commercial orchard conditions, in disseminating the pathogen and, possibly, contributing to disease spread. A pollen lot, certified free from Psa, was used with the same methods as a control. Two pollination techniques were used: dusting (dry pollen) and spraying (pollen suspension in water). The orchard was monitored during 2 years from experimental pollination, with regular sampling of flowers, fruits, leaves, and vines, to check for Psa as an epiphyte or endophyte, and for bacterial canker symptoms. Psa was recovered from flowers, fruitlets and leaves during the first season, mainly in plots where contaminated pollen had been sprayed in water suspension. From early August until harvesting time (mid-October), Psa detection was possible only on leaves. No symptoms developed during the first season after pollination. No endophytic Psa was detected in pruned vines in the following winter. During the second season, detection and isolation of Psa was erratic, but direct isolation was achieved from four plots. During the second season after pollination, typical leaf symptoms were observed on a few vines, and Psa was isolated and identified. Our results suggest that Psa could be disseminated via contaminated kiwifruit pollen as a pathway for spread of bacterial canker. However, further pollination experiments are needed to establish, beyond any doubt, whether contaminated pollen may contribute to possible disease outbreaks

Isolation of bacterial endophytes from Actinidia chinensis and preliminary studies on their possible use as antagonists against Pseudomonas syringae pv. actinidiae

Abstract:BACKGROUND:The bacterial canker, caused by Pseudomonas syringae pv. actinidiae, is the most severe disease of cultivated Actinidia spp. The pathogen is systemic and not easily controlled by agrochemical means. OBJECTIVE:Our aim was to search, select and identify, among kiwifruit bacterial endophytes, possible antagonists able to control Pseudomonas syringae pv. actinidiae. METHODS:Several kiwifruit production areas were inspected, in order to find host plants without any disease symptom inside severely affected orchards. From those plants, endophytes were isolated, selected, tested for their ability to inhibit the growth of the pathogen and identified. RESULTS:A set of 65 different bacterial endophytes was isolated and tested: several of them were able to inhibit the growth of Pseudomonas syringae pv. actinidiae in vitro. None of the antagonists proved to possess either ice nucleation activity or transferable copper resistance. Taxonomically, antagonists belonged to the families of Pseudomonadaceae and Enterobacteriaceae. CONCLUSIONS:Effective bacterial antagonists were found as endophytes in kiwifruit plants and bearing features of safety of use and negligible risk for the crop. Such finding makes it possible to select the most prospective of them, in order to develop efficient biopesticides able control the bacterial canker in commercial orchards

The epiphitic life of Pseudomonas syringae pv. actinidiae on kiwifruit and other cultivated and spontaneous plants.

Bacterial canker of kiwifruit is the most destructive disease of cultivated Actinidia spp. The causal agent is the Gram-negative bacterium Pseudomonas syringae pv. actinidiae. Several surveys have been carried out in kiwi orchards in Romagna (northern Italy) and proved that Psa may have a significant epiphytic life, mainly on flowers, pollen and leaves. On the fruits of affected orchards Psa was detectable until early August. In Romagna, kiwifruit is cultivated in areas where also other fruit crops are grown, often side by side. A two-year survey confirmed that Psa is present as a leaf epiphyte in neighbouring stone fruit orchards (apricot and plum), when an affected kiwi orchard is nearby. Several other spontaneous plants and weeds present in kiwifruit orchards have been checked for the epiphytic presence of Psa. Stinging nettle was constantly associated with the epiphytic presence of Psa, thus providing good conditions for bacterial growth and survival. Psa was also detected on, or isolated from other weeds, like amaranth, common mallow, purslane, dandelion and smartweed. The time range during which Psa was detected/isolated on other plants was the same as Psa could be traced on kiwi plants. These results suggest that Psa, like other pathogenic and non pathogenic pseudomonads, might spend a significant lapse of time on non-host plants. The role in the epidemiology of the bacterial canker of kiwifruit of such epiphytic population is not known. As a good practice for kiwifruit orchards, control of weeds, especially in case of Psa presence, is particularly advised

Endophytes and epiphytes from Actinidia spp. as potential agents for the biocontrol of the bacterial canker of kiwifruit.

The bacterial canker of kiwifruit is the most destructive disease of cultivated Actinidia spp. The causal agent is the Gram- negative bacterium, Pseudomonas syringae pv. actinidiae (Psa). The two commonly grown host species, Actinidia deliciosa (green fleshed) and Actinidia chinensis (yellow fleshed) show different susceptibility to the disease: the yellow kiwifruit is far more susceptible than the green one. The pathogen has significant epiphytic and endophytic phases: it penetrates the host through natural openings and thorough wounds. The control of the disease is difficult, due to the lack of effective chemicals that might be applied in the orchards. Copper compounds alone did not yield a satisfactory control of the disease. For endophyte and epiphyte isolation, orchards were chosen where the disease had the highest incidence and severity. Inside orchard plots, where most kiwi plants were dead, occasional symptomless plants were observed, cut and taken to the lab. Epiphytes were isolated and purified from washings of the leaves and endophytes were obtained from the washings of woody disks, taken at different levels along the trunk. A selection of ca. 60 isolates was chosen and subjected to preliminary genetic characterisation with rep-PCR, using the BOX primer. The selected endophytes and epiphytes were assayed in vitro for their ability to inhibit a set of phytopathogenic Gram-negative and Gram-positive bacteria, among them Psa. The results of the experiments showed that at least a dozen of endophytes and epiphytes were able to strongly inhibit both Psa and other important phytopathogenic bacteria

ISOLATION AND CHARACTERIZATION OF AN ENDO- PHYTE FROM ACTINIDIA SP. SHOWING A STRONG ANTAGONISTIC ACTIVITY AGAINST PSEUDOMONAS SYRINGAE PV. ACTINIDIAE

The bacterial canker of kiwifruit is the most destructive disease of cultivated Actinidia spp. The causal agent is the Gram negative bacterium Pseudomonas syringae pv. actinidiae (Psa). The pathogen grows inside the trunk, vines and leaves of its host plants, causing wilting and formation of cankers, with subsequent death of the plant. During the past three years, samples have been collected from various parts of asymptomatic Actinidia spp. within infected kiwi orchards and many endophytic bacteria have been isolated. Among them, a few isolates were identified and proved to be able to strongly inhibit Psa namely an isolate of Pantoea agglomerans, two pseudomonads belonging to the fluorescens/putida group, and one novel Pseudomonas sp., which proved to be highly effective in inhibiting, in vitro, several important phytopathogenic bacteria. This last isolate was chosen for further studies. Concentrated supernatant of its liquid culture in LB medium could inhibit Psa, indicating that the active compound produced by the antagonist is excreted from the cell into the environment. The nature of that biotoxin is not proteic, and its size is smaller than 3 kDa, as a result of its partial purification. Moreover, a bank of mutants deficient in their antagonistic activity has been constructed by triparental mating, in order to search and identify genes involved in antagonism against Psa. Fifty six prospective mutants have been already obtained: twenty two of them were subject to Southern blotting to verify a single transposon insertion. Gene identification is under way

Molecular characterisation of an endophyte showing a strong antagonistic activity against Pseudomonas syringae pv. actinidiae

Abstract Background and aims Endophytic bacteria have been often studied as biological control agents of plant pathogens and many of their secondary metabolites involved in antagonism are Non Ribosomal Peptides (NRPs). In this study, the molecular basis of the biocontrol properties of the endophyte Pseudomonas synxantha, isolated from Actinidia chinensis, against Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial kiwi canker, has been investigated. Methods Antagonism-deficient mutants of P. synxantha strain DLS65 were generated by insertion of minitransposon mTn5-GNm in its genome. Southern blot analysis allowed the selection of single transposon insertion-mutants. Amplification of the transposon flanking regions by means of arbitrary and single primer PCR in selected mutants was perfomed to obtain amplicons for sequencing purposes. Results Sequencing results of the amplicons obtained from three antagonism-deficient mutants led to the localization of the transposonin three genes, which implies their involvement in the antagonism of P. synxantha: an acyl-homoserine lactone acylase gene (pvdQ), a glucose-6-phosphate dehydrogenase gene (zwf) and an mbtH-like gene were identified. It is known from the literature that these three genes are involved directly or indirectly in NRPs synthesis. Conclusions We suggest that a molecule with antibiotic properties, produced by NRP synthetases, contributes to the antagonistic activity of P. synxantha. Keyword