© 2019 Vieira dos Santos M.C., Horta J., Moura L., Pires D.V., Conceição I., Abrantes I., Costa S.R.The nematophagous fungus Pochonia chlamydosporia, a natural enemy of plant parasitic nematodes (PPN), has been exploited in the development of sustainable management strategies for PPN control. The intrinsic variation among P. chlamydosporia isolates affects biocontrol potential, so sound characterisation is required. Biological, molecular and metabolic analyses can be determinant in the screening and selection of these potential biological control agents. This study aimed to provide integrative characterisation of P. chlamydosporia isolates that can support isolate selection for biological control purposes. Eight Portuguese isolates, associated with Meloidogyne spp., were used as a case study. Isolates were identified and characterised: i) at the biological level through standard bioassays to evaluate their ability for rhizosphere colonisation, to produce chlamydospores, and to parasitise Globodera pallida and Meloidogyne incognita eggs; ii) at the molecular level, examining genetic variation using ERICPCR; and iii) at the metabolic level, assessing their metabolic profiles using Biolog FF MicroPlates, with concurrent reads of fungal utilisation of 95 different carbon sources. Molecular data and metabolic characterisation were reduced using Principal Component Analysis and compared with the biological characteristics. Molecular profiles could only be related to isolate geographical origin, but the original substrate (eggs or roots), parasitism of M. incognita eggs and ability for rhizosphere colonisation were correlated with metabolic profiles, indicative of utilisation of specific carbon sources. Meloidogyne egg parasitism and rhizosphere colonisation were related to each other. This integrative characterisation offers novel perspectives on the biology and biocontrol potential of P. chlamydosporia, and, once tested on a broader set of isolates, could be used to assist rapid isolate selection.

© International Society for Horticultural Science. All rights reserved.Bacterial canker of kiwifruit, caused by Pseudomonas syringae pv. actinidiae (Psa), is a disease included on the EPPO A2 List because it is currently increasing in the Mediterranean region. Climatic conditions determine the multiplication and dispersal behavior of Psa and may influence its biological control (BC). However, information regarding the influence of climatic conditions on growth of the Psa bacterium in Portugal is scarce. The aims of this study were to relate the climatic conditions with the efficacy of Bacillus subtilis Serenade Max® in controlling the disease, to determine whether disease incidence increased over the growing season, and to monitor carry-over of disease from one season to the next. Two ‘Hayward’ orchards of Actinidia deliciosa of different ages, located in Valença, in the north of Portugal, were selected for BC assay. Thirty plants were randomly selected from a young orchard (6 years old) and another 30 plants from an old one (30 years old). Psa was identified and characterized by morphological, biochemical and molecular tests. The efficacy of B. subtilis was evaluated by disease incidence, and correlated to climatic conditions. A standardized leaf infection index (0-4 scale of symptoms) was used to determine disease severity. Monitoring of Psa symptoms was performed every 30 days, from June 2016 to April 2017. The severity of the disease decreased in the summer season, characterized by unfavorable conditions for Psa such as high temperatures and scarce precipitation. B. subtilis was partially effective in controlling Psa in the 6-year-old orchard, and showed no efficacy in the protection of leaves in the 30-year-old orchard. B. subtilis applied at flowering could be considered as a suitable solution for the control of Psa biovar 3 only for the new kiwifruit orchard (6 years old).

© 2018 The Society for Applied Microbiology Aims: Bacterial kiwifruit canker disease, caused by Pseudomonas syringae pv. actinidiae (Psa) was detected in north-west Portugal in 2010, and has since caused significant losses. The objectives of this work were to characterize the Portuguese population(s) of Psa and to define the actual prevalence of Psa biovars in the most productive kiwifruit region in Portugal. Methods and Results: Isolates obtained from Actinidia deliciosa orchards were characterized by morphological, biochemical, physiological, fatty acids and molecular tests (PCR, BOX-PCR, duplex-PCR, multiplex-PCR and RFLP), phaseolotoxin, housekeeping and effector genes and pathogenicity. Results established that only Psa biovar 3 is present in the north-west of Portugal, despite phenotypic and genetic variability among the isolates. Conclusions: This work provides new information on P. syringae pv. actinidiae (Psa) genetic profile in Portugal, indicating for the first time, that two genetically different subpopulations of Psa biovar 3 are present. Significance and Impact of the Study: A new subpopulation of Psa biovar 3 was found for the first time in Portugal, contributing to increase knowledge about this population worldwide and to support further understanding of the impact of Psa.

Pseudomonas corrugata and P. mediterranea are non fluorescent Pseudomonas species that represent major groups of bacteria responsible for tomato pith necrosis. P. viridiflava is also reported as an important pathogen associated with this disease. The aim of the present study was to determine differences in abilities of P. corrugata, P. mediterranea, Pmc (a new Portuguese lineage of P. corrugata) and P. viridiflava to colonize tomato roots and stems from contaminated soil. One strain representative of each species was chosen after a characterization of a collection of Portuguese pathogenic strains using phenotypic and molecular tools (Moura, 2005). Following inoculation of the soil substrate, presence (using Bio-PCR) and population sizes of the different bacterial strains were monitored in soil substrate, root and stem fragments at regular sampling dates (3, 9, 16, 28, 37 and 49 days after inoculation). Strains of P. corrugata, P. mediterranea and Pmc were always detected in the soil substrate during seven weeks following inoculation, with a concentration decreasing from 3, 44 x 108 to 6, 97 x 104 ufc g -1 of substrate. The behaviour of these three pathogens was similar concerning their progression inside tomato tissues, with population sizes ranging from 9, 98 x 105 to 1, 64 x 104 ufc g-1 root. These three strains progressed into tomato stems ahead of the soil until the fourth internode in the absence of disease symptoms. P. viridiflava was not detected in contaminated soil substrate seven weeks after inoculation. It did not survive in the roots for a long time, and did not progress in tomato's stem. In conclusion, P. corrugata and its variant Pmc, and P. mediterranea were able to colonize tomato roots and stems from contaminated soil without plant injury. In contrast, P. viridiflava was not able to maintain detectable populations in soil neither to colonize tomato plants from contaminated soil.

A polymerase chain reaction (PCR) method that combines biological and enzymatic amplification (BIO-PCR) is described for the detection of fluorescent and non-fluorescent Pseudomonas, associated with tomato pith necrosis in naturally infected plants under commercial greenhouse conditions, in the north and south of Portugal. Sections from the basal, median and apical parts of tomato stems were macerated with water and aliquots of the extracts were plated onto selective agar media. After 24 h of incubation, the plates were washed to remove bacterial cells and aliquots were analyzed by PCR, without prior DNA extraction. Primers PC1/1, PC1/2, PC5/1 and PC5/2 were used in a multiplex PCR for the detection of Pseudomonas corrugata and P. mediterranea. Primers Pvir1 and Pvir2 were used in a standard PCR for the detection of P. viridiflava. BIO-PCR detected P. corrugata in the north, P. mediterranea in the south and P. viridiflava in both regions. Overall results suggested that BIO-PCR may be a useful technique for routine detection of these pathogens under field conditions, as well as for epidemiological and host-pathogen interaction studies.