First Report of Clonostachys rhizophaga as a Pathogen of Dendrocalamus giganteus in Mozambique.

Dendrocalamus giganteus Wall. ex Munro, a wild plant belonging to the family Poaceae, is widespread in Mozambique where it is used as a construction material. At the end of 2007, disease symptoms have been observed on D. giganteus plants growing in the neighborhood of Maputo. Diseased plants showed longitudinal dark streaks on the stem surface to which corresponded internal vascular browning and chlorosis in wide leaves that gradually developed into necrosis. At the final stage of the disease plants died. To isolate the pathogen, stem segments collected during September 2008 were surface sterilized with 1% HgCl2 for 30 s, rinsed with sterile deionized water for 30 s, and incubated on potato dextrose agar (PDA) medium at 22°C in the dark for 2 weeks. Monosporic cultures of the isolated fungus formed dimorphic Verticillium-like (primary) or penicillate (secondary) conidiophores and ovoidal to elongate, minutely curved, hyaline conidia, 5 to 9.5 × 2.5 to 4.5 µm, with laterally displaced hilum. These characteristics are typical of Clonostachys rhizophaga Schroers (3). Identification was confirmed by the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands) on the basis of the ß-tubulin (tub2) gene sequence (3). For our isolate CBS 125416, the tub2 sequence was 100% similar to that of the C. rizhophaga strain CBS 124511 (GenBank Accession No. FJ 593883) (1). To verify the pathogenicity of the fungus, a 5-mm-diameter mycelial plug obtained from 2-week-old colonies grown on PDA was affixed to a portion of the stem of D. giganteus from which the superficial tissues had been removed and the inoculation site was covered with wet cotton and wrapped with Parafilm. Control plants were treated by the same method but using PDA plugs without mycelium. Twenty plants were used, ten of which were controls. They were grown in a controlled climatic chamber at 22°C with a photoperiod of 16 h at 40 µE·m-2·s-1. Two months after inoculation, all plants showed a dark area surrounded by an idropic halo on the stem surface and internal browning, whereas control plants remained healthy. C. rhizophaga was recovered from all infected plants. C. rhizophaga is apparently rare. The fungus (as Verticillium rhizophagum Tehon & Jacobs, nom. invalid.) has been previously reported from the United States, Chile, and Ecuador (4) and as a culture contaminant in Switzerland (3). More recently C. rhizophaga has been found to be associated with Pinus canariensis in Argentina (2) and it has been reported as a causal agent of chickpea wilt in Syria (1). To our knowledge, this is the first report of C. rhizophaga for subsaharian Africa. It may be under reported and more common than has been thought because of the difficulty in identifying Clonostachys species, but with the ability to identify species using tub2 (3), there can be no doubt of its identity. References: (1) M. M. Abang et al. Plant Dis. 93:666, 2009. (2) L. Eduardo Piontelli and G. Giusiano. Bol. Micol. 18:89, 2003. (3) H. J. Schroers. Stud. Mycol. 46:85, 2001. (4) L. R. Tehon and H. L. Jacobs. Bull. Davey Tree Expert Company, Kent, OH. 6:3, 1936.

First Report of Downy Mildew Caused by Plasmopara helianthi Races 700 and 703 of Sunflowers (Helianthus annuus) in Italy.

Sunflower (Helianthus annuus L.) plants showing systemic downy mildew (Plasmopara helianthi Novot.) symptoms (stunting and leaf chlorosis) were collected during May and June 2001-2003 in fields located in four regions of central Italy (Umbria, Tuscany, The Marches, and Emilia-Romagna). Generally restricted, a low disease incidence (2 to 12%) was observed in fields planted with commercial hybrids. In the surveyed areas, higher field incidences (20 to 30%) of downy mildew were only observed where suitable climatic conditions occurred. To determine pathogen race variability of P. helianthi, 18 isolates (one for each field) were recovered directly from infected leaves showing sporulation or after 24 h of incubation in a humid chamber at room temperature. After increasing initial inocula on the susceptible cv. Ala, race identification of all isolates was determined by the whole-seedling immersion technique (2) on three sets of nine differential sunflower lines (three lines per each set): HA-304, RHA 265, RHA 274; PMI-3, PMI-17, 803-1; and HAR-4, QHP-1, HA 335 using triplet coding (1). All differential lines were tested twice with 40 seeds per replicate (three replicates per line). Twelve days after inoculation, plants were placed in a chamber maintained at 20°C and 100% relative humidity for 24 h and then evaluated for a susceptible (sporulation on cotyledons and/or first true leaves) or resistance (absence of sporulation) reaction. Races 700 and 703 were identified during the 3-year survey. Race 700 was recovered frequently (56% of isolates) from the Italian regions except Emilia-Romagna. Race 703 was not detected in Tuscany. Races 700 and 703 have been previously isolated in other European countries (3,4), but to our knowledge, this is the first report of the occurrence of either race in Italy. Our results confirm previous investigations and suggest that the restricted presence of downy mildew should be attributed to several factors: (i) lack of any source of resistance to both races in commercial hybrids; (ii) insufficient seed dressing with metalaxyl as observed on remnants of commercial hybrid treated seeds tested in laboratory analyses; and (iii) short rotation of cereals (often only 2- to 3-year interval) with sunflower crops. Although greenhouse tests showed most P. helianthi isolates were not controlled by metalaxyl seed treatment at the registered rate (2.1 g kg-1) loss of fungicide efficiency in fields has not yet been observed indicating that appropriate phytosanitary measures and proper seed treatment can provide control of sunflower downy mildew. References: (1) T. J. Gulya. Pages 76-78 in: Proc. 17th Sunflower Res. Workshop, Fargo, ND, 1995. (2) T. J. Gulya et al. Helia 14:11, 1991. (3) M. L. Molinero-Ruiz et al. Plant Dis. 86:736, 2002. (4) F. Virany and T. J. Gulya. Plant Pathol. 44:619, 1995.

First Report of Puccinia helianthi (Race 314) on Sunflower in Mozambique.

Rust of sunflower (Helianthus annuus L.) caused by Puccinia helianthi Schwein. is a major sunflower disease in Mozambique where local ecotypes, from Peredovik and Record selections, and South African commercial varieties were all susceptible to race 4, which was prevalent among isolates collected from 1996 to 1997 (1). Disease surveys carried out in southern Mozambique (Maputo) from August until September 2002 revealed an increase in the incidence and severity of sunflower rust compared to previous investigations on the same varieties. Rust symptoms, observed in all fields, appeared on the last leaf pairs of plants at the 5.1 growth stage. Disease severity (percentage of leaf area infected) (2) ranged from 20 to 60%, but the disease did not cause noticeable yield loss because rust infection started near the end of the season. Argentine and African commercial varieties and some experimental Mozambican selections from sunflower accessions of different origin were the most severely affected. Two rust samples were collected from infected leaves, stored at 5°C, and increased on the universal suscept (S-37-388). Race identification of both isolates was determined on three sets of nine differential lines (three lines per each set): S-37-388, CM 90 RR, MC 29; P-386, HA-R1, HA-R2; and HA-R3, HA-R4, HA-R5 using the triplet coding system (3). The 2-week-old seedlings (four per 10-cm pot) of each differential line (four pots per line) were inoculated on the upper surface of the first pair of true leaves. Inoculated seedlings were covered with plastic bags, placed in a growth chamber without light for 18 to 22 h, and then transferred to a greenhouse under alternate day/night temperatures of 22/18 ± 2°C, 60 to 70% relative humidity, and 12 h of light (180 µE·m-2·s-1) per day. The inoculation test was repeated twice. Assessment of host differential reactions, 14 days after inoculation, was based on five categories of sunflower reaction to rust (4). Both isolates were identified as race 314 and to our knowledge, this is the first report of this race in Mozambique. Since P. helianthi completes its life cycle annually, the occurrence of new races is expected. The opportunity for new races to occur is particularly high and severe in Mozambique where all sunflower varieties grown as summer (January to June) and winter (August to December) crops are susceptible. References: (1) P. M. Vicente and A. Zazzerini. Helia 20:25, 1997. (2) T. J. Gulya et al. NDSU Extension Serv. PP-998, 1990. (3) T. J. Gulya and S. Masirevic. FAO Eur. Res. Network on Sunflower. Bucarest, Romania, 31, 1995. (4) S. M. Yang. Ann. Phytopathol. Soc. Jpn. 52:248, 1986.

Behavior of metalaxyl and its pure R-enantiomer in sunflower plants (Helianthus annus).

A possible stereospecific and/or stereoselective mechanism of biodegradation for metalaxyl and metalaxyl-M was studied to elucidate their behavior in sunflower plants and to compare their biodegradation. Greenhouse experiments were carried out to confirm the same efficacy of the two fungicides against infections by Plasmopara helianthi in sunflower plants. The two fungicides appear to have the same behavior regarding both the protection against plant infections and the mode of translocation and the rate and pathway of biotransformation, but we have evidence that this biotransformation process is enantioselective. Furthermore, we propose procedures for a chromatographic separation of enantiomers and acid metabolites of the fungicides and for the determination of the R:S ratio by HPLC chiral analyses. This study emphasizes the importance of examining the fate of both stereoisomers of a chiral agrochemical in an environmental system for the correct use of enantiomerically pure agrochemical compounds.

Isolation and characterization of phytotoxic compounds produced by Phomopsis helianthi.

The isolation, chemical characterization and biological activity of two phytotoxic metabolites of Phomopsis helianthi Munt-Cvet et al. is reported. These compounds were identified by spectroscopic methods (UV, IR, 1H and 13C NMR, and MS) as trans-4,6-dihydroxymellein (trans-3-methyl-4,6,8-trihydroxy-3,4-dihyroisocoumarin) and cis-4,6-dihydroxymellein (cis-3-methyl-4,6,8-trihydroxy-3,4-dihydroisocoumarin). This is the first report of the isolation of trans-4,6-dihydroxymellein from fungal cultures and of the production of cis- and trans-4,6-dihydroxymelleins by P. helianthi. Rice was found to be a good substrate for the production of the dihydroxymelleins. Culture extracts of some Italian and French strains of P. helianthi showed different degrees of phytotoxicity towards sunflower leaves and seedlings. The minimum effective doses of trans- and cis-4,6-dihydroxymelleins with different bioassays were 76 and 135 microg per spot (leaf puncture bioassay), 3 and 5 micromol g(-1) fresh tissue (absorption by leaf cutting) and 5 and 2 micromol g(-1) fresh tissue (absorption by cut seedlings), respectively. These compounds may contribute to the severity of the sunflower disease caused by P. helianthi.

First Report of Downy Mildew (Race 2) of Sunflowers in Italy.

During a survey in the spring of 1997, sunflower (Helianthus annuus L.) plants with characteristic symptoms (stunting and veinal chlorosis of leaves) of downy mildew caused by Plasmopara helianthi Novot. were observed in two experimental fields in the central Italian region of Umbria. In the first field, used to evaluate 58 commercial sunflower hybrids, fewer than 1% of plants displayed typical downy mildew symptoms. In a second field, planted with the old susceptible cultivar Ala, disease incidence was 12%. The inoculum of P. helianthi (one isolate from each field) was obtained either from downy mildew-infected leaves or infested soil (from around the roots of diseased plants). All were collected according to a random distribution. From this collection, two isolates were identified as race 2 by the whole-seedling immersion technique (2) with differential cultivars: HA 89, AD 66, CM 90 RR, RHA 265, RHA 274, HIR 34, DM-2, and HA 335. All differential lines were tested twice with 30 seeds per replicate (four replicates per line). Two weeks after inoculation the percentage of susceptible seedlings (showing sporulation on cotyledons and/or first true leaves) was determined. Race 2 has long occurred in some European countries, but this is the first report of this race from Italy. Extensive field surveys carried out in the last 10 years revealed that race 1 is the only one occurring in central Italy. An isolate apparently comparable to race 2 was reported in experimental plots in 1975 (1) where only Pl1 cultivars were susceptible. Differential lines, at that time, were not available and race identification was not carried out. Italy imposes importation regulations for sunflower seed that include treatment with metalaxyl. In greenhouse trials we have not observed a reduction in sensitivity to this systemic fungicide used as seed treatment against P. helianthi race 2. Consequently, the susceptibility of some commercial hybrids, indicated as resistant to downy mildew by importers, could be attributed to irregularity in the production of seeds. Also, an insufficient seed dressing with metalaxyl does not completely control the pathogen. References: (1) M. D'Armini et al. Atti Giornate Fitopatol. 1:659, 1975. (2) T. J. Gulya et al. Helia 14:11, 1991.

First Report of Root-Knot Nematodes (Meloidogyne spp.) on Sunflowers in Mozambique.

Sunflower (Helianthus annuus L.) recently was reintroduced to Mozambique because of renewed interest in oil-seed production for domestic consumption. In April 1997, disease surveys were carried out in two fields in southern Mozambique (Maputo region). Several plants of Pan 735, a South African cultivar, showed yellowing of the leaves and stunting. These plants wilted during the day but recovered their turgidity at night. Diseased plants were easily pulled from the soil due to almost complete destruction of the root system. Numerous galls were found on affected roots, compared with healthy plants. Meloidogyne javanica (Treub) Chitwood and M. incognita (Kofoid & White) Chitwood were identified by M. Di Vito (personal communication) based on 20 female perineal patterns observed with a light microscope. M. incognita was more prevalent than M. javanica. Also observed were Alternaria helianthi (Hansf.) Tubaki & Nishihara and Sclerotium bataticola Taub. Root-knot nematodes (Meloidogyne spp.), common on sunflower, cause severe damage and reduce both seed yield and seed oil content (1). These two nematode species have also been observed on sunflower in other African countries (Zambia, South Africa, Egypt) but this is the first report of root-knot nematode on sunflower in Mozambique. Reference: (1) M. Di Vito et al. Nematol. Medit. 24:109, 1996.