Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.

AIMS: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus more costly and time-consuming, or using only one fluorescent dye, thus less accurate. METHODS AND RESULTS: A red primary fluorescence (autofluorescence) of the hyphal cell walls and septa of Rhizoctonia spp. with green excitation is evidenced in Rhizoctonia spp. This property is exploited and combined for the first time with a conventional DAPI fluorescence to accurately determine the nuclear condition of Rhizoctonia. This bi-fluorescence imaging strategy depicted the nuclear condition in Rhizoctonia spp. more accurately than the conventional DAPI fluorescence used alone and was validated against isolates previously genotyped by DNA sequencing. CONCLUSIONS: We demonstrated that the bi-fluorescence imaging strategy was safe, accurate and simple to perform and interpret. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed bi-fluorescence imaging strategy provides a sensitive tool for determining the nuclear condition of Rhizoctonia strains. Its simplicity is a key advantage when there are numerous cultures to be examined.

First Report of Black Swallow-Wort as an Alternate Host of the Two-Needle Pine Stem Rust Pathogen, Cronartium flaccidum, in France.

Stem rust disease, caused by Cronartium flaccidum (Alb. & Schwein.) G. Winter, is among the most destructive diseases of the two-needle hard pine in the Northern Hemisphere, including Scots pine but also several Mediterranean pines in southern Europe (2,3). This heteroecious rust has numerous alternate herbaceous hosts spanning different plant families, thereby contributing to epidemic outbreaks when environmental conditions for infection are optimal (2,3). The main alternate host in Europe is the white swallow-wort, Vincetoxicum hirundinaria Medik, a herbaceous perennial in the milkweed family (Asclepiadaceae). At the southwestern edge of its distribution, V. hirundinaria co-occurs with the black swallow-wort, V. nigrum (L.) Moench and cases of misidentification between the two species are not uncommon. Little to no disease occurs to V. nigrum likely because phenanthroindolizidine alkaloid antimicrobial compounds are produced in the weed. In 1918, occurrence of C. flaccidum was reported in Spain and Portugal on black and white swallow-worts albeit as C. asclepadium (1). In the early summer of 2011, at Saint Clément de Rivière in southern France, we detected orange-yellow rust pustules on the lower leaf surfaces of several black swallow-worts growing near Aleppo pines (Pinus halepensis). These orange-yellow pustules were erumpent uredinia in groups (range = 137 to 400 µm in diameter) with peridia that broke with the production of uredinospores. The latter were moderately echinulate, light yellow, broadly ellipsoid (length = 23 ± 4 µm and range = 11 to 33 µm; width = 15 ± 2 µm and range = 9 to 20 µm) with walls of 1 to 2 µm thick (mean 1.3 ± 0.2 µm). Hair-like columnar telia (length = 1123 ± 131 µm and range = 976 to 1280 µm; width = 136 ± 28 µm and range = 104 to 176 µm) were mostly formed from uredinia. Telia were hypophyllous and reddish orange. Teliospores were orange-yellow and ellipsoidal to cylindrical (length 26.3 ± 6.2 µm and range 13.5 to 46 µm; width = 10.5 ± 1.8 µm and range = 6.9 to 14.9 µm). Morphological features of these fruiting structures were consistent with those of C. flaccidum (Alb. & Schwein.) G. Winter on white swallow-worts (2). Additional confirmation was provided by sequencing the two internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S gene (4). The sequence was 843 bp long (GenBank Accession No. JN802139), 99.7% similar to C. flaccidum found on Melampyrum in Finland (Accession No. JF13709), and 99.4% similar to C. flaccidum found on pines in Italy (Accession No. X83900). Voucher material has been deposited at the Herbarium of Montpellier's University under the collection Accession No. MPUØ188846. To our knowledge, this is the first report of the occurrence of uredinia and telia of C. flaccidum on black swallow-worts clearly identified in France. The occurrence of the rust on this understory vine is of critical importance for the economic sustainability of pine forests in France, especially when they are heavily constrained by drought and fire. References: (1) R. Gonzalez Fragoso. Trab. Mus. Nac. Ci. Nat., Ser. Bot. 15:1, 1918. (2) J. Kaitera and H. Nuorteva. For. Pathol. 33:205, 2003. (3) A. Ragazzi. Phytopathol. Medit. 28:5, 1989. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

Foliar and Cane Rot of Arundo donax Caused by Nigrospora oryzae in Europe.

A fungus was isolated consistently from dead shoot tips and flag leaves of Arundo donax L. (Poaceae) in France, Crete, Cyprus, Italy, Morocco, and Spain from April of 2003 through September of 2005. The fungus was identified as Nigrospora oryzae (Berk. & Br.) Petch (teleomorph Khuskia oryzae) on the basis of morphological characteristics (1). The mean diameter of 80 conidia obtained from sporulating plant specimens collected in France, Crete, and Cyprus were 14, 15, and 15 µm, respectively. The mean diameters of 25 conidiogenous cells and conidiophores were 7 and 4 µm, respectively. Identification was confirmed by comparing the sequence of the ribosomal DNA internal transcribed spacer 1 and 4 regions from the French isolate (GenBank Accession No. DQ219433) with the sequence of a voucher specimen from the Museum National d'Histoire Naturelle, Paris, France. The isolate of N. oryzae from France was deposited at the CBS collection in Utrecht, the Netherlands (CBS 113884). N. oryzae is known to be a weak pathogen on a wide range of plants but has not been reported on A. donax, which is now a well-established weed in the United States and North America, probably originating from the Mediterranean Region. Herein, the possible use of N. oryzae as a biological control agent was investigated. Twenty young A. donax shoots growing in the greenhouse and 20 emerging canes in the field were selected on the basis of uniformity in size. A spore suspension in distilled water adjusted to 5 × 105 conidia/ml of the French isolate was prepared and 0.5 ml was injected with a syringe just below the growing point of the flag leaf in onehalf of the greenhouse and field plants. The remaining plants were injected with 0.5 ml of distilled water as controls. Infection and death of the flag leaf occurred in 30% of the shoots in the greenhouse and 50% of the canes in the field 21 days from inoculation. No disease developed on the control plants. Greenhouse inoculation tests were repeated once. N. oryzae was reisolated from dead leaves and the terminal node of inoculated shoots, satisfying Koch's postulate. Attempts made to induce disease symptoms by applying spore suspensions on the whorl of leaves surrounding the apical tip failed. This is an indication that an insect vector may be needed to carry and deposit N. oryzae spores into the tight, whorled flag leaf for infection and disease development to occur. To our knowledge, this is the first report of foliar and cane rot of A. donax caused by N. oryzae. References: (1) K. H. Domsch et al. Page 515 in: Compendium of Soil Fungi. IHW-Verlag, Eching, Germany, 1993.

Factors Affecting Infection of Yellow Starthistle (Centaurea solstitialis) by Synchytrium solstitiale, Causal Agent of False Rust Disease.

Yellow starthistle (Centaurea solstitialis) is an annual invasive weed in the United States with Mediterranean origins. The expense of chemical control and the vast area of invasion make this weed an appropriate target for classical biological control. Observations of a field site in southern France revealed small orange galls on the leaves of yellow starthistle seedlings caused by the fungus Synchytrium solstitiale. Inoculation of yellow starthistle seedlings with a suspension of zoospores released from infected tissue resulted in infection. Ten days after inoculation, typical orange galls appeared on the exposed tissue. Preliminary host range testing showed up to 100% infection of C. solstitialis seedlings from both France and the United States and infection of Carthamus tinctorius, Centaurea americana, C. diffusa, C. rothrockii, C. squarrosa, and Helianthus annuus seedlings. No symptoms were observed on seedlings of Centaurea calcitrapa, C. maculosa, C. sulfurea, Cirsium californica, C. occidentale, Cynera cardunculus, and Taraxacum officinale. Zoospores were released in a pH range between 4.45 and 8.25 and optimally at temperatures between 5 and 15°C. Infection of yellow starthistle seedlings occurred after a minimum 1-h exposure to a zoospore suspension at 20°C.