First Report of Twig Blight Disease of Citrus Caused by Haematonectria haematococca in the Philippines.

Citrus has recently become one of the most important crops in the Philippines with approximately 151,000 t of production in 2005. A new disease affected citrus (Citrus reticulata Blanco cv. Satsuma) twigs and trunks in 2005. The problem has caused significant concerns to the industry because of its destruction and high severity of infection. Symptoms include twig drying, bark hardening, twig girdling, leaf chlorosis, and defoliation. Infection occurs at all stages of the plant where severe symptoms were observed from January to April. Average temperature of these months ranged from 20 to 28°C. Affected twigs were often covered with pink or salmon-colored fungal propagules (1). In April 2009, citrus twigs displaying the above-described symptoms and signs were collected from Malabing Valley, Kasibu, Nueva Vizcaya, the Philippines (16°20'4.6912″N, 121°16'41.0742″E). Fungal perithecia were surface sterilized and observed with a stereomicroscope. Single ascospore was picked and inoculated into the peptone-pentachloronitrobenzene medium (2) and the plates were incubated at 22 ± 2°C for 7 days with a 12-h light/dark regimen. Periodically, the plates were screened for the growth of mycelia or discrete colonies on the medium. The cultured fungus produced microconidia and multiple canoe-shaped macroconidia. With a hand-held sprayer, approximately 20 ml of distilled water containing fungal conidia (2 × 105 spores/ml) was inoculated each onto 1-year-old cv. Satsuma citrus (30 plants) bearing young twigs. Control plants were sprayed with sterile distilled water. After inoculation, plants were maintained at 20 to 28°C and 75 to 85% relative humidity, enclosed in clear plastic bags, placed under 70% woven shade cloth, and watered regularly. The inoculated plants started showing the initial twig blight symptoms in all inoculated branches at 37 days post inoculation (dpi). The occurrence of pink-to-orange perithecia of Haematonectria haematococca was observed at 45 dpi, which has similar morphological appearance to the perithecia collected from the field. Symptoms were not observed on branches treated with sterile water. H. haematococca was reisolated from the symptomatic twigs and the recovered isolates were morphologically similar to H. haematococca, thus completing Koch's postulates. Control plants remained healthy. DNA was extracted from all isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 and sequenced. A portion of the ITS sequence has been deposited in the NCBI database (GenBank Accession No. HQ696788). A BLAST search of the NCBI database with the ITS sequence revealed H. haematococca (Berk. & Broome) Samuels & Nirenberg as the closest match with 100% sequence similarity. To our knowledge, this is the first report of H. haematococca causing citrus twig blight in the Philippines. To maintain the economic profitability of the citrus industry in the Philippines, control measures must be implemented to minimize tree loss. References: (1) P. J. Chester. Phil. Agri. Rev. 11:69, 1919. (2) S. M. Nash and W. C. Snyder. Phytopathology 52:567, 1962.

A New Biotype of Phaeosphaeria sp. of Uncertain Affinity Causing Stagonospora Leaf Blotch Disease in Cereals in Poland.

A new Phaeosphaeria sp. biotype was isolated from winter ryes in Poland during 1995. Two isolates, Sn23-1 and Sn48-1, were obtained from diseased leaves of cvs. Motto and Dankowskie, respectively. The rye Phaeosphaeria sp. represented by isolate Sn48-1 has similar pycnidiospore morphology and induces disease symptoms in cereals similar to Phaeosphaeria nodorum, the causal agent of Stagonospora nodorum blotch disease (4). The pathogen (Sn48-1) produces hyaline, cylindrical pycnidiospores that are mostly three-septate and measure 12.8 to 23.7 × 2.1 to 3.2 µm (average size = 16 × 2.6 µm) on water agar. A molecular comparison of several genes in isolates Sn23-1 and Sn48-1 revealed that the rye Phaeosphaeria sp. was different from P. nodorum. In the conserved alpha-box sequence (1,93 bp) of the MAT1-1 gene, a four nucleotide difference occurred between the wheat-biotype P. nodorum and isolates Sn23-1 and Sn48-1 (GenBank Accession Nos. AY072933 and AF322008). In addition, the length of the internal transcribed spacer (ITS) region of the nuclear rDNA was the same for the wheat-biotype P. nodorum and the two rye Phaeosphaeria sp. isolates. However, a six nucleotide discrepancy was found in the ITS region (GenBank Accession Nos. U77362 and AF321323). The beta-glucosidase (bgl1) and beta-tubulin (tubA) genes differ in length between the wheat-biotype P. nodorum and two rye Phaeosphaeria sp. isolates (2,3). The main difference was due to the intron sizes of these two genes. One extra nucleotide was found in the intron2 of the bgl1 gene (GenBank Accession Nos. AY683619 and AY683620) and the intron1 of the tubA gene (GenBank Accession Nos. AY786337 and AY786331), respectively, in these two rye Phaeosphaeria sp. isolates. Disease severity on the fifth leaf (GS15) of Polish wheat (Alba, Begra, and Liwilla), triticale (Bogo and Pinokio), and rye (Zduno) cultivars was assessed with one (resistant) to nine (susceptible) scales 14 days after inoculation. Aggressiveness of wheat-biotype P. nodorum isolate Sn26-1 and rye Phaeosphaeria sp. isolate Sn48-1 was significant (P < 0.01) in five cultivars except in the moderately resistant wheat cv. Liwilla. The rye Phaeosphaeria sp. isolate Sn48-1 severely affected Polish rye Zduno (8.3) and two triticale cultivars (6.5), while the infection by isolate Sn26-1 was moderate (3-4). On the contrary, the wheat-biotype P. nodorum isolate Sn26-1 was more aggressive on wheat (4.1 on moderately resistant Alba and 6.2 on highly susceptible Begra) than the rye Phaeosphaeria sp. isolate Sn48-1, which had a scale of 2.2 and 4.3, respectively. Under laboratory conditions, the rye isolate Sn48-1 was able to cross with the wheat-biotype P. nodorum isolate Sn26-1 that has an opposite mating-type (MAT1-2) gene, but few viable ascospores were produced (1). References: (1) P. C. Czembor and E. Arseniuk. Mycol. Res. 104:919, 2000. (2) A. Malkus et al. FEMS (Fed. Eur. Microbiol. Soc.) Lett. 249:49, 2005. (3) E. Reszka et al. Can. J. Bot. 83:1001, 2005. (4) M. J. Richardson and M. Noble. Plant Pathol. 19:159, 1970.

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae.

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.

A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae.

Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.

Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae.

The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.

SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms.

Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes.

Genetics of Host Specificity in Epichloë typhina.

ABSTRACT Epichloë typhina perennially and systemically infects grass plants, causing choke disease in which maturation of host inflorescences is suppressed. In seedling-inoculation tests, isolate E8 from perennial ryegrass established and maintained infection in this host but not in orchardgrass. In contrast, isolates E469, E2466, and E2467 from orchardgrass varied in infection frequency and stability in orchardgrass, but all were unable to establish stable infections in perennial ryegrass. To investigate the genetics of host specificity, isolate E8 was crossed with each of the isolates from orchardgrass. Seedlings of parental host species were inoculated with F(1) progeny, and the frequencies of seedling infection and stability in adult plants were assessed. In the E8 x E2466 cross, the F(1) progeny exhibited a wide range of infection frequency and stability in each parental host. In crosses E8 x E469 and E8 x E2467, where the orchardgrass-derived parents infected 5 to 13% of inoculated perennial ryegrass seedlings, the distributions of infection frequencies for the F(1) progeny wereskewed toward levels comparable to that of the parent from perennial ryegrass. In all crosses, most progeny had low frequencies of infection in orchardgrass. However, transgression was evident in a cross of E8 with E469, an isolate that infected orchardgrass seedlings at a low frequency (2 to 3%). The E8 x E469 cross had a few F(1) progeny that infected orchardgrass at high efficiency (up to 81%). A Spearman rank correlation applied to the E8 x E2466 progeny indicated a significant negative correlation between infection frequencies in perennial ryegrass and orchardgrass. Also, there was a significant correlation of infection frequency and stability in perennial ryegrass but not in orchardgrass. To test whether only a few genes governed infection frequency in perennial ryegrass, an E8 x E2466 F(1) progeny (designated E386.04), which had intermediate compatibility with this host, was backcrossed to E8. The progeny of this backcross exhibited a distribution of infection frequencies in perennial ryegrass between that of E386.04 and the backcross parent, suggesting that multiple genes may determine compatibility at the seedling infection stage. The results of these experiments indicated multiple genetic determinants of compatibility or incompatibility with each host, with intermediate or high heritability.

Inheritance of mitochondrial DNA and plasmids in the ascomycetous fungus, Epichloë typhina.

We analyzed the inheritance of mitochondrial DNA (mtDNA) species in matings of the grass symbiont Epichloë typhina. Eighty progeny were analyzed from a cross in which the maternal (stromal) parent possessed three linear plasmids, designated Callan-a (7.5 kb), Aubonne-a (2.1 kb) and Bergell (2.0 kb), and the paternal parent had one plasmid, Aubonne-b (2.1 kb). Maternal transmission of all plasmids was observed in 76 progeny; two progeny possessed Bergell and Callan-a, but had the maternal Aubonne-a replaced with the related paternal plasmid Aubonne-b; two progeny lacked Callan-a, but had the other two maternal plasmids. A total of 34 progeny were analyzed from four other matings, including a reciprocal pair, and in each progeny the plasmid transmission was maternal. The inheritance of mitochondrial genomes in all progeny was analyzed by profiles of restriction endonuclease-cleaved mtDNA. In most progeny the profiles closely resembled those of the maternal parents, but some progeny had nonparental mtDNA profiles that suggested recombination of mitochondrial genomes. These results indicate that the fertilized stroma of E. typhina is initially heteroplasmic, permitting parental mitochondria to fuse and their genomes to recombine.

The Fusarium solani gene encoding kievitone hydratase, a secreted enzyme that catalyzes detoxification of a bean phytoalexin.

Among the antimicrobial phytoalexins produced by Phaseolus vulgaris (French bean) is the prenylated isoflavonoid, kievitone. The bean pathogen, Fusarium solani f. sp. phaseoli, secretes a glycoenzyme, kievitone hydratase (EC 4.2.1.95), which catalyzes conversion of kievitone to a less toxic metabolite. Among F. solani strains, those that are highly virulent to P. vulgaris also produce kievitone hydratase constitutively, suggesting that the enzyme is a virulence factor. Based on the N-terminal amino acid sequence of purified enzyme, the kievitone hydratase cDNA and gene (khs) were cloned. The identities of khs and the cDNA were confirmed by their expression in transgenic Neurospora crassa and Emericella nidulans. Based on the gene and cDNA sequences, khs is predicted to encode a preprotein of 350 amino acids, from which a 19 amino acid N-terminal transit peptide is removed during maturation and secretion. The predicted mass of the mature polypeptide, 37 kDa, contrasts with the 47 to 49 kDa size estimated by electrophoresis of purified enzyme, confirming that the enzyme is extensively glycosylated. The inferred polypeptide sequence has seven canonical sites for N-glycosylation. Southern blot-hybridization analysis of F. s. f. sp. phaseoli DNA indicates one khs locus and an additional locus with weak hybridization to the khs probe. Sequences related to khs were also detected in several isolates of F. solani and the related teleomorph, Nectria haematococca. However, strains of F. oxysporum known to exhibit inducible kievitone hydratase activity (but not pathogenic to bean) did not have detectable khs homology. Nevertheless, all isolates known to cause severe disease on bean possessed khs sequence.