Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

Characterisation of a novel ilarvirus causing grapevine angular mosaic disease

Unusual symptoms were observed on ‘Baresana’ x ‘Baresana’ Vitis vinifera hybrid vines in the Grapevine Variety Collection of the Grapevine Institute, Athens. The affected vines showed sharp angular mosaic on leaves, along the veins and in vein angles, malformations, abortive flowers or very few berries with smaller, wrinkled and non-germinating seeds, as well as gradual decline, severe stunting and death of the vine. Serological tests on diseased vines for the presence of 13 known grapevine viruses gave negative results. An infectious agent was transmitted mechanically to several herbaceous indicator plants. Koch’s Postulates were fulfilled, and the agent, proven to be a virus, was named Grapevine angular mosaic virus (GAMV). Serological tests have been developed for the virus. The most conserved polymerase region showed significant similarity of GAMV with members of subgroup 1 of the Ilarvirus genus; however ML phylogenetic analysis could not support its clustering within this subgroup. GAMV differs serologically and in particle morphology from Grapevine line pattern virus (GLPV) a putative member of the Ilarvirus genus that infects grapevine. It is proposed that GAMV is a novel member of the Ilarvirus genus.     

<Emphasis Type="Italic">Fusarium mangiferae</Emphasis> associated with mango malformation in the Sultanate of Oman

Mango malformation, caused by Fusarium mangiferae, represents the most important floral disease of mango. The first symptoms of this disease were noticed in the beginning of 2005 in plantations at Sohar in the Sultanate of Oman. The affected inflorescences were abnormally enlarged and branched with heavy and dried-out panicles. Based on morphology and DNA-sequence data for the genes encoding translation elongation factor 1α and β-tubulin, the pathogen associated with these symptoms was identified as F. mangiferae.     

Leaf spot of tobacco caused by <Emphasis Type="Italic">Fusarium proliferatum</Emphasis>

In 2014 and 2015, an unknown leaf spot disease was found on tobacco in Guangxi, China. The fungus isolated from these spots was identified as Fusarium proliferatum based on morphological characteristics and sequence analysis of translation elongation factor 1 alpha (tef1α). This fungus also reproduced leaf spot symptoms after inoculation and was reisolated from the symptomatic lesions. This is the first report of a new leaf spot caused by Fusarium proliferatum on tobacco.     

Multiplex PCR assay for simultaneous detection of MBI-D and Q<Subscript>o</Subscript>I resistance in rice blast fungus

For sustainable control of rice blast with fungicides, efficient monitoring of the emergence and spread of fungicide-resistant isolates is needed. We developed simple and reliable PCR-based DNA markers to detect isolates resistant to melanin biosynthesis inhibitor targeting scytalone dehydratase (MBI-D) and quinone outside inhibitor (Q_oI) fungicides. Through the use of DNA templates prepared from mycelia on filter paper or from infected leaves, these markers enable rapid (a few hours) genotyping of point mutations that confer resistance. The developed multiplex marker detected resistance to both MBI-D and Q_oI in a single PCR and further reduced the time needed for diagnosis.     

Five plant families support natural sporulation of <Emphasis Type="Italic">Cronartium ribicola</Emphasis> and <Emphasis Type="Italic">C. flaccidum</Emphasis> in Finland

Fusarium is one of the most destructive fungal genera whose members cause many diseases on plants, animals, and humans. Moreover, many Fusarium species secrete mycotoxins (e.g. trichothecenes and fumonisins) that are toxic to humans and animals. Fusarium isolates from date palm trees showing disease symptoms, e.g. chlorosis, necrosis and whitening, were collected from seven regions across Saudi Arabia. After single-sporing, the fungal strains were morphologically characterized. To confirm the identity of morphologically characterized Fusarium strains, three nuclear loci, two partial genes of translation elongation factor 1 α (tef1α) and β-tubulin (tub2), and the rDNA-ITS region, were amplified and sequenced. Of the 70 Fusarium strains, 70 % were identified as F. proliferatum that were recovered from six regions across Saudi Arabia. Fusarium solani (13 %), as well as one strain each of the following species: F. brachygibbosum, F. oxysporum, and F. verticillioides were also recovered. In addition, five Fusarium-like strains were recognized as Sarocladium kiliense by DNA-based data. The preliminary in vitro pathogenicity results showed that F. proliferatum had the highest colonization abilities on date palm leaflets, followed by F. solani. Although F. oxysporum f. sp. albedinis is the most serious date palm pathogen, F. proliferatum and F. solani are becoming serious pathogens and efforts should be made to restrict and control them. In addition, the potential toxin risks of strains belonging to F. proliferatum should be evaluated.     

Pathogenicity of plant and soil isolates of <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> on tomato and pepper

Crown and root rot of tomato and sweet pepper can be caused by Phytophthora parasitica. In this work, 23 P. parasitica isolates from diseased pepper or tomato plants as well as 54 isolates from 23 monocrop tomato soils (from Spain and Chile) and one from a pepper soil were studied for their host–pathogen response. Results show significant host specificity for the isolates from tomato plants and tomato soils (63 of 64 isolates were unable to cause disease in pepper). None of the pepper plant/soil isolates showed pathogenicity on tomato, and only four of 14 reproduced their pathogenicity on pepper. Only one tomato isolate was pathogenic to both Solanaceae species. Two different inoculation protocols were evaluated (substrate irrigation and stem cutting). All isolates which expressed pathogenicity when stem inoculated also did it when root inoculated, but not vice-versa. Therefore, the recommended test protocol for tomato and pepper breeding programmes is that based on root inoculation by irrigation.     

Differential expression of molecular rust resistance components have distinctive profiles in <Emphasis Type="Italic">Coffea arabica</Emphasis> - <Emphasis Type="Italic">Hemileia vastatrix</Emphasis> interactions

Countering the economic hurdle caused by coffee leaf rust disease is most appealing at this time as it has posed a major threat to coffee production around the world. Establishing differential expression profiles at different times following pathogen invasion in both innate and acquired immunities unlocks the molecular components of resistance and susceptibility. Suppression subtractive hybridization (SSH) was used to identify genes differentially over-expressed and repressed during incompatible and compatible interactions between Coffea arabica and Hemileia vastatrix. From 433 clones of expressed sequence tags (ESTs) sequenced, 352 were annotated and categorized of which the proportion of genes expressed during compatible interaction were relatively smaller. The result showed upregulation and downregulation of various genes at 12 and 24 h after pathogen inoculation in both interactions. The use of four different databases in searching for gene homology resulted in different number of similar sequences. BLASTx against EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics Institute) database being with the maximum (100%) hits for all the annotated sequences. RT-qPCR analysis of seven resistance-signaling genes showed similar expression patterns for most of the genes in both interactions, indicating these genes are involved in basal (non-specific) defense during which immune reactions are similar. Using SSH, we identified different types of resistance related genes that could be used for further studies towards resistant cultivar development. The potential role of some of the resistance related proteins found were also discussed.     

Recovery of fungicide-resistant <Emphasis Type="Italic">Botrytis</Emphasis> isolates from strawberry nursery plants

In a four-year survey of strawberry nursery plants, 340 Botrytis isolates were collected and examined for fungicide resistance. High percentages of isolates with resistance to members of all fungicide classes registered on strawberries in Germany were found, i.e. trifloxystrobin (90.3%), boscalid (53.8%), cyprodinil (41.5%), fludioxonil (28.2%) and fenhexamid (15.6%). Of these isolates, 10.3% possessed multiple resistance to all compounds. At 5.0%, resistance to the recently registered fungicide fluopyram was low. Dutch nursery material harboured significantly higher proportions of isolates with resistance to any or all fungicides than plants produced in Germany. These data point to nursery plants as a possible route of introducing fungicide-resistant Botrytis strains into commercial strawberry fields.