Pantoea agglomerans as a New Etiological Agent of a Bacterial Necrotic Disease of Mango Trees.

Bacterial apical necrosis of mango trees, a disease elicited by Pseudomonas syringae pv. syringae, is a primary limiting factor of mango crop production in the Mediterranean region. In this study, a collection of bacterial isolates associated with necrotic symptoms in mango trees similar to those produced by bacterial apical necrosis disease were isolated over five consecutive years in orchards from the Canary Islands. The bacterial isolates were characterized and identified as Pantoea agglomerans. Pathogenicity tests conducted on onion bulbs and mango plants confirmed that P. agglomerans strains isolated from mango trees are a new etiological agent of a bacterial necrotic disease in the Canary Islands. Pathogenicity plasmids of the pPATH family have been previously reported in P. agglomerans. The majority of putatively pathogenic (n = 23) and pathogenic (n = 4) P. agglomerans strains isolated from mango trees harbored four plasmids, one of which was close in size to the 135-kb pPATH pathogenicity plasmid. The analysis of the presence of two major genes in pPATH plasmids (repA and hrpJ) was undertaken in P. agglomerans strains isolated from mango trees. The hrpJ gene was detected in the 140-kb plasmid of pathogenic P. agglomerans strains isolated from mango trees but it showed differences in nucleotide sequences compared with other pathogenic strains. In contrast, the repA gene was not detected in any of the putatively pathogenic and pathogenic P. agglomerans strains isolated from mango trees. Finally, genetic characterization and phylogenetic analysis using the hrpJ gene and the housekeeping genes gyrB and rpoB showed that almost all P. agglomerans strains that were putatively pathogenic and pathogenic on mango trees clustered together, forming a differentiated phylogroup with respect to the other pathogenic P. agglomerans strains described from other hosts.

Resistance to the SDHI Fungicides Boscalid, Fluopyram, Fluxapyroxad, and Penthiopyrad in Botrytis cinerea from Commercial Strawberry Fields in Spain.

Gray mold, caused by the necrotrophic fungus Botrytis cinerea., is one of the most economically important diseases of strawberry. Gray mold control involves the application of fungicides throughout the strawberry growing season; however, B. cinerea isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish gray mold population. Succinate dehydrogenase inhibitors (SDHI) constitute a relatively novel class of fungicides registered for gray mold control representing new alternatives for strawberry growers. In the present study, 37 B. cinerea isolates previously characterized for their sensitivity to boscalid and amino acid changes in the SdhB protein were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to fluopyram, fluxapyroxad, and penthiopyrad. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 580 B. cinerea isolates collected from 27 commercial fields in Spain during 2014, 2015, and 2016. The EC50 values ranged from 0.01 to >100 µg/ml for fluopyram, <0.01 to 4.19 µg/ml for fluxapyroxad, and, finally, <0.01 to 59.65 µg/ml for penthiopyrad. Based on these results, as well as findings from a previous publication, the discriminatory doses chosen to examine sensitivities to boscalid, fluopyram, fluxapyroxad, and penthiopyrad were 100, 15, 1, and 6 µg/ml, respectively. Over the course of the 3-year monitoring period, the overall frequencies of resistance to the four SDHI were 56.9, 6.9, 12.9, and 24.6%, respectively. The frequency of boscalid-resistant isolates decreased from 73 to 41% over the years; however, the fluopyram-resistant isolates increased from 5 to 10% after 1 year of registration. Four SDHI resistance patterns were observed in our population, which included patterns I (30%; resistance to boscalid), II (13.8%; resistance to boscalid and penthiopyrad), III (5.7%; boscalid, fluxapyroxad, and penthiopyrad), and IV (7.9%; resistance to boscalid, fluopyram, fluxapyroxad, and penthiopyrad). Patterns I and II were associated with the amino acid substitutions H272R and H272Y; pattern III was associated only with the H272Y mutation; and, finally, pattern IV was associated with the N230I allele in the SdhB subunit. For gray mold management, it is suggested that the simultaneous use of boscalid and penthiopyrad should be limited to one application per season; however, fluxapyroxad and, especially, fluopyram could be used as valid SDHI alternatives for gray mold control, although they should be applied with caution.

Further Support of Conspecificity of Oak and Mango Powdery Mildew and First Report of Erysiphe quercicola and Erysiphe alphitoides on Mango in Mainland Europe.

Mango leaves and inflorescences infected by powdery mildew in southern Spain were analyzed using multigene sequencing (ITS + 4 single-copy coding genes) to identify the causal agent. Erysiphe quercicola was detected in 97% out of 140 samples, collected in six different orchards in the Malaga region. Among these, a small proportion also yielded E. alphitoides (8% of all samples) and E. alphitoides was found alone in 3% of samples. A phylogenetic approach was completed by cross inoculations between oak and mango, which led to typical symptoms, supporting the conspecificity of oak and mango powdery mildews. To our knowledge, this is the first report of E. quercicola and E. alphitoides causing powdery mildew on mango trees in mainland Spain, and thus mainland Europe, based on unequivocal phylogenetic and biological evidence. Our study thus confirmed the broad host range of both E. quercicola and E. alphitoides. These results have practical implications in terms of the demonstrated ability for host range expansion in powdery mildews. They also open interesting prospects to the elucidation of molecular mechanisms underlying the ability to infect single versus multiple and unrelated host plants since these two closely related powdery mildew species belong to a small clade with both generalist and specialist powdery mildews.

Characterization of Resistance to Six Chemical Classes of Site-Specific Fungicides Registered for Gray Mold Control on Strawberry in Spain.

Botrytis cinerea, causal agent of the gray mold disease, is one of the most economically important fungal pathogens of strawberry worldwide. In Spain, as in other parts of the world, management of gray mold control primarily involves the application of fungicides. To determine the fungicide resistance of the Spanish strawberry field population, 367 B. cinerea isolates were examined from one organic and 13 conventional strawberry fields in Huelva (Spain) in 2014 and 2015. The sensitivities of these isolates to six fungicides used for gray mold management in Spain were examined using a spore germination assay based on previously published discriminatory doses. The frequency of resistance to pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil was 74.6, 64.8, 37.0, 23.7, 14.7, and 0.8%, respectively. The majority of isolates (35.1%) were resistant to three different fungicides classes. Within these isolates, the most prevalent resistance profile (55.8%) was resistance to pyraclostrobin, boscalid, and cyprodinil, followed by the resistance profile (30.2%) of resistance to pyraclostrobin, boscalid, and fenhexamid. One isolate collected in 2015 was resistant to all six fungicide classes. Resistance to boscalid, fenhexamid, iprodione, and pyraclostrobin was found to be caused by amino acid substitutions on target proteins, including H272R/Y in SdhB, F412I/S/V in Erg27, I365 N/S in Bos1, and G143A in Cytb, respectively. The presence of multifungicide resistance phenotypes in B. cinerea isolates from strawberry fields in Spain must be considered in the development of future resistance management practices.

Transient transformation of Podosphaera xanthii by electroporation of conidia.

BACKGROUND: Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew. RESULTS: To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B. CONCLUSION: This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.

The Podosphaera fusca TUB2 gene, a molecular "Swiss Army knife" with multiple applications in powdery mildew research.

The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii) is the main causal agent of cucurbit powdery mildew and one of the most important limiting factors for cucurbit production worldwide. Despite the fungus' economic importance, very little is known about the physiological and molecular processes involved in P. fusca biology and pathogenesis. In this study, we isolated and characterised the ß-tubulin-encoding gene of P. fusca (PfTUB2) to develop molecular tools with different applications in powdery mildew research. PfTUB2 is predicted to encode a protein of 447 amino acid residues. The coding region is interrupted by six introns that occur at approximately the same positions as the introns present in other fungal TUB2-like genes. Once cloned, the PfTUB2 sequence information was used in different applications. Our results showed that the TUB2 gene is a good marker for molecular phylogenetics in powdery mildew fungi but it is unsuitable for the analysis of intraspecific diversity in P. fusca. The expression of PfTUB2 was proven to be stable in different temperature conditions, supporting its use as a reference gene in quantitative gene expression studies. Furthermore, an allele-specific PCR assay for the detection of resistance to methyl-2-benzimidazole carbamate (MBC) fungicides in P. fusca was developed based on the correlation between the single amino acid change E198A in ß-tubulin and the MBC resistance phenotype. Lastly, PfTUB2 was used as a target gene in the development of a high-throughput method to quantify fungal growth in plant tissues.