DIAGNOSIS IN PHYTOBACTERIOLOGY 2 – Pseudomonas spp. in stone fruits Dr. Jaap D. Janse Department Laboratory Methods and Diagnostics Dutch General Inspection Service (NAK) Emmeloord, The Netherlands [email protected] COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas amygdali - Bacteriosis or Bacterial hyperplastic canker of almond • Reported from Greece and Turkey. Italy at risk • Only on almond, perennial cankers • Forgotten pathogen , produces t-zeatin and IAA, genes could be used for detection/idenitfication www.atlasplantpathogenicbacteria.it COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas avellenae - Canker or decline of hazelnut • Reported from Greece in 1976, later in central Italy (2001). • Turkey at risk • In some years and some areas devastating • Two lineages (Greece and Italy) with very different type III secreted effectors (T3SE’s). Molecular dating: divergence predates observation in the field – epidemic possibly caused by change in agricultural practice. COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse 1 Pseudomonas avellenae - Canker or decline of hazelnut • Related bacteria P.s. pv. coryli and P.s. pv. syringae , causing bacterial twig dieback of hazelnut in C. Italy and Sicily and S. Germany COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas avellenae - Canker or decline of hazelnut Detection and identification • primers PAV 1 and PAV 2 with 762-bp product Scortichini & and Marchesi (2001) J. Phytopathol. 149:527-532. • hrpW-derived primers, with 350 bp product Loreti & Gallelli (2002), Eur. J. Plant Pathol. 108: 237-244 • Pathogenicity test, needle inoculation of leaf scars of young trees, may take up to 7 months • Biochemical tests • ANI study – confirms species status . Scortichini M, Marcelletti S, Ferrante P, Firrao G (2013) A Genomic Redefinition of Pseudomonas avellanae species. PLoS ONE 8(9): e75794. doi:10.1371/journal.pone.0075794 COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas avellenae - Canker or decline of hazelnut Biochemical tests Test P avellanae P.s pv coryli Pss Pmorsprun Ice - V + - nucleation Utilization of - - + - adonitol sorbitol - + + + erythritol - - + V L+tartrate - - - + L-lactate - - + - Gelatine - - + - liquefaction Arbutin - + + - hydrolysis Aesculin - + + V hydrolysis syrB gene - - + - COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse 2 Pseudomonas syringae pv. avii - Canker of wild cherry • Reported from France, 2003. Very much related to Pss . Also pathogenic to cultivated cherry. Pseudomonas syringae pv. avii (pv. nov.), the Causal Agent of Bacterial Canker of Wild Cherries (Prunus avium) in France, Menard et al., 2003 European Journal of Plant Pathology 2003, 109: 565-576 • Strains from Psa , Pss and Pmp studied in Belgium (A. Bultreys, M. Steenackers) • Will be discussed with Pss COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae pv. castaneae - Canker of chestnut (Castanea) • Reported from Japan in 1989 from Castanea crenata • Cankers formed in spring on twigs that cause bud death and leaf blight and small galls on shoots, petioles, midveins and flower clusters • Related to P. syringae pv. eriobotryae , causing stem canker of loquat ( Eriobotrya japonica – Rosaceae) Forgotten disease • Kazuo & Kanji (1989) Annals Phytopath. Soc. Japan 55: 397- 403 COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae pv. morsprunorum - Bacteria canker of stone fruits, blossom blast, gummosis, leaf spot of stone fruits • Canker phase on shoots, branches and limbs most important. • Leaf scar infection and dispersal by rain splash. Hail storms favor the leaf spot phase. • Two races (based on phage typing), some host specificity, discriminated with rep-PCR. Ecological significance? www.atlasplantpathogenicbacteria.it COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse 3 Pseudomonas syringae pv. persicae (EPPO Quarantine List A2) - Dieback/canker on nectarine and peach • First observed in France 1967 and proved to be present also in New-Zealand in 1988 where also Prunus salicina (Japanese plum) was infected. • Restricted occurrence in France, Germany and Portugal . Reported from Croatia and United Kingdom. Only sporadic outbreaks Photo’s : INRA Luisetti, France COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae pv. persicae (EPPO Quarantine List A2) - Dieback/canker on nectarine and peach • EPPO standard PM 7/43 • Biochemical tests • Pathogenicity test in dormant 1 year-old shoots of young trees from mid September to the end of January). • No DNA tests available (see EFSA Scientific Opinion on the pest categorisation of Pseudomonas syringae pv. persicae (Prunier et al.) Young et al. EFSA Journal 2014;12(10):3855 (26pp) Not used • Produces persicomycins, phytotoxic compounds may yield tool for PCR detection Barzic (1999) Physiol. Mol. Plant Path. 55: 243-250 COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae pv. syringae - Bacteria canker or blast of stone and pome fruit, apoplexy of apricot, dead bud of cherry, twig dieback in hazelnut • Pss has many hosts, very diverse pathovar, many strains show some host specificity • INA-positive, associated with (night) frosts plum Courtesy M. Sulikowska, PL Courtesy D. Burokienė Lithuania www.atlasplantpathogenicbacteria.it COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse 4 Pseudomonas syringae pv. syringae - Bacteria canker or blast of stone and pome fruit, apoplexy of apricot, dead bud of cherry, twig dieback in hazelnut Symptoms on sweet cherry in USA Source: Kari Peter, Ph.D. Department of Plant Pathology and Environmental Microbiology Penn State University Fruit Research and Extension Center Biglerville, PA [email protected] COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae pv. syringae - Bacteria canker or blast of stone and pome fruit, apoplexy of apricot, dead bud of cherry, twig dieback in hazelnut Predisposing factors in USA (K. Peter) favoring the occurrence of bacterial canker • Sandy and clay soils • Nutrient deficiency • High ring nematode populations • Winter pruning • Severe winter freezes • Spring freezes • Ice nucleation – intercellular liquid freezes at higher temperatures – cell damage – leaking – bacteria multiply • injured plant tissue COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Predisposing factors in USA (K. Peter) favoring the occurrence of bacterial canker (continued) • Bacteria overwintering in buds, cankers • P. syringae populations increase 10 – to 100 – fold during bloom (blossom infection – blossom blast) • Summer: – Humid, wet weather: symptoms on leaves and fruit; Hot and dry conditions: P. syringae populations low • Autumn rains and cooler temperatures: P. syringae detected at high levels prior to and during leaf fall -Infection at leaf scars can be high COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse 5 Pseudomonas syringae pv. syringae - Bacteria canker or blast of stone and pome fruit, apoplexy of apricot, dead bud of cherry, twig dieback in hazelnut • Pss for first time in Irak in 2014 on almond, plum and peach but not on cherry Hassan et al., 2014 IOSR Journal of Agriculture and Ve terinary Science 7:1-8 • But in Iran in 2014 also on sweet cherry Host specificity, pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts. Rezaeia & Taghavia Archives of Phytopathology and Plant Protection 47, 2014 COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae pv. syringae - Bacteria canker or blast of stone and pome fruit, apoplexy of apricot, dead bud of cherry, twig dieback in hazelnut Hypothetical life cycle of P. syringae indicating important inoculum sources from the environment (air water) for the orchards Source: The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. Morris et al., The ISME Journal (2008) 2, 321–334 COST FA1104 Training School Molecular diagnostics of bacterial diseases, Zürich, Switzerland, 2015-09-21-25 – Jaap D. Janse Pseudomonas syringae General methods – well developed by Belgian lab, Alain Bultreys • Hildebrands pectate medium Kings medium B KBC boric acid and fungicides MS3 in Latvian lab Vicente et al. (2004) 110: 337-351 • Ice nucleation Lindow 1990 Cortesy A. Bultreys B • Toxin detection (lipodepsipeptides) biological test on PDA the yeast Geotrichum candidum