Toxigenic capacity and trichothecene production by Fusarium graminearum isolates from Argentina and their relationship with aggressiveness and fungal expansion in the wheat spike.

At least 20 epidemics of Fusarium head blight (FHB) of wheat have been registered in the last 50 years in Argentina, with variable intensity. Damage induced by the disease is further aggravated by the presence of mycotoxins in affected grains that may cause health problems to humans and animals. The trichothecene chemotype was analyzed for 112 isolates of Fusarium graminearum from Argentina by polymerase chain reaction and two field trials were conducted to study the aggressiveness of a subsample of 14 representative isolates and to analyze deoxynivalenol (DON) production in planta and in vitro. All isolates belonged to the 15-acetyl-DON chemotype. Significant differences were observed in both the symptom severity induced in wheat spikes and the in vivo DON production, and a close correlation was found between these two variables. However, in vitro toxigenic potential was not correlated with the capacity of F. graminearum isolates to produce DON under natural conditions. The progress of infection in the rachis of inoculated wheat spikes was analyzed and the pathogen presence verified in both symptomatic and symptomless spikes. Even isolates with a limited capacity to induce symptoms were able to colonize the vascular tissue and to produce considerable amounts of DON in planta.

Fusarium Wilt of Lettuce Caused by Fusarium oxysporum f. sp. lactucae in Argentina.

The green belt area surrounding the city of La Plata, Argentina, produces more than 70% (around 280 ha) of the lettuce (Lactuca sativa L.) cultivated under greenhouse for fresh consumption in the country. In February 2011, April 2012, and December 2013, butterhead lettuce plants from cv. Lores showing wilt and stunted growth symptoms, red-to-brown discoloration of vascular tissues, and yellow leaves were found in greenhouses in La Plata. Sections of tap root, crown and stem from symptomatic plants showing dark-brown streaking of the vascular tissue were surface sterilized and isolations were made. A total of 21 monosporic isolates obtained from different lettuce production fields were identified as Fusarium oxysporum Schltdl. based on morphological characteristics (2). Vegetative compatibility group (VCG) analysis (1) was conducted on 11 of these isolates and all isolates belonged to the same VCG. To fulfil Koch's postulates, two pathogenicity tests were conducted with these 11 isolates in winter (July 2012) and summer (December 2013). Healthy 20-day-old butterhead lettuce seedlings of two cultivars (Reina de Mayo and Lores in the winter and summer tests, respectively) were inoculated by dipping the roots of each plant in a spore suspension (~3 × 105 CFU ml-1), planted in 1-liter pots containing autoclaved soil and grown in a greenhouse with only natural daylight. Control treatments were prepared by dipping the seedling roots in sterilized distilled water. All inoculated plants showed wilt symptoms 15 to 20 days after inoculation (dai) and 45 to 50 dai in the summer and winter pathogenicity tests, respectively. The delay in the appearance of symptoms observed during the winter test is consistent with the effect of planting date on the development and final incidence of Fusarium wilt of lettuce reported by Matheron et al. (3). No symptoms were observed in control treatments. F. oxysporum was re-isolated from vascular tissues of the stems of symptomatic plants and the formae speciales lactucae J.C. Hubb. & Gerik was confirmed by PCR using the specific primer pair GYCF1 and R943 (4). The identification of only one VCG for the tested isolates agrees with the hypothesis of seed transmission of the pathogen, which might explain the dissemination of Fusarium wilt of lettuce in geographically distant areas (2). Studies are being carried out to determine the race of these Argentinian isolates of F. oxysporum f. sp. lactucae. To our knowledge, this is the first report of F. oxysporum f. sp. lactucae infecting lettuce in Argentina. References: (1) J. C. Correll et al. Phytopathology 77:1640, 1987. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) M. E. Matheron et al. Plant Dis. 89:565, 2005. (4) G. C. Y. Mbofung and B. M. Pryor. Plant Dis. 94:860, 2010.

First Report of Fusarium Wilt of Basil Caused by Fusarium oxysporum f. sp. basilici in Argentina.

Annually, ~20 ha of sweet basil (Ocimum basilicum L.) are cultivated in greenhouses in the green belt area surrounding La Plata, Argentina, mainly for fresh consumption. In 2004 to 2007, basil plants of cv. Genovese showed wilt symptoms, necrosis of leaves and stems, asymmetrical growth, and discolored vascular tissue in greenhouses in La Plata. In 2007, the same symptoms were observed on plants of cv. Morada grown from seeds that were produced in Italy. Isolations were completed from root, crown, and stem sections of diseased plants of cv. Genovese from three greenhouses in 2004 to 2007, and from commercial seeds, stem sections, flowers, and seeds of diseased plants of cv. Morada in 2007. Seeds and portions of symptomatic tissues were surface-disinfested with 0.5% NaOCl for 1 min, rinsed in sterilized distilled water, air dried, and plated on 2% potato dextrose agar (PDA). Twenty-seven isolates were identified as Fusarium oxysporum Schltdl. based on morphological characteristics (4), and the species identification confirmed by PCR assay using a F. oxysporum f. sp. basilici-specific primer pair, Bik 1 and Bik 2 (1). Vegetative compatibility groups (VCGs) were determined for the 27 isolates through complementation of nitrate-nonutilizing mutants generated from these isolates (2) and paired with two Italian tester strains from an international collection (PVS-Fu 220 and PVS-Fu 125, provided by V. Balmas, Univeristà degli Studi di Sassari, Italy). All 27 isolates from Argentina belonged to VCG 0200. This is a unique VCG for F. oxysporum f. sp. basilici and has been identified in Israeli, American, and Italian isolates of the fungus (3). To fulfill Koch's postulates, pathogenicity tests were conducted with 12 isolates selected to reflect the multiple sources of fungal recovery, including root, crown, and stem sections, and leaves of diseased plants of cv. Genovese and commercial seeds, stem sections, flowers, and seeds of cv. Morada. Isolates were each grown on moistened (40% w/w), autoclaved, polished rice for 10 days, dried, and ground in a grinder. The number of CFU/g rice was determined by serial dilution plating onto PDA plates. The inoculum was added to autoclaved soil at 104 CFU/g dry soil. For each isolate, 8 healthy basil seedlings of each of cvs. Genovese and Morada were planted in pots, each containing 1 liter of inoculated soil. The control treatment consisted of 8 basil seedlings of each of the same cultivars planted in autoclaved soil mixed with sterilized, ground, polished rice. Plants were grown in a greenhouse with natural daylight for 45 to 50 days after inoculation. All inoculated plants showed the same symptoms described for the original basil plants. No symptoms were observed on the control plants. F. oxysporum f. sp. basilici was re-isolated from the vascular tissue of stems of symptomatic plants but not from control plants, and species identification confirmed by PCR assay as previously described. The presence of the pathogen was verified in the seed lot produced in Italy, suggesting that this could have been a source of inoculum that introduced the pathogen into La Plata, Argentina, as supported by the hypothesis that infested seed resulted in spread of a clonal population of F. oxysporum f. sp. basilici internationally (1). To our knowledge, this is the first report of F. oxysporum f. sp. basilici infecting sweet basil in Argentina. References: (1) A. Chiocchetti et al. Plant Dis. 85:607, 2001. (2) J. C. Correll et al. Phytopathology 77:1640, 1987. (3) A. Garibaldi et al. Plant Dis. 81:124, 1997. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

Basal Rot of Hylocereus undatus Caused by Fusarium oxysporum in Buenos Aires, Argentina.

Night-blooming cereus (Hylocereus undatus A. Berger) is generally used as rootstock of ornamental cactus because of its rapid growth and tolerance to humid substrates. Since 2002, in Gran Buenos Aires, a new disease has been observed in grafted crops in all production stages. Incidence was as much as 30% in many greenhouses. Symptoms consisted of soft rot that started near the soil line and developed upward until it affected all the rootstock. The scion was not rotten, but died as a consequence of rootstock infection. All the roots were symptomless. For pathogen isolation, symptomatic tissues were surface disinfected by a 1-min immersion in 0.2% NaOCl, placed on potato dextrose agar (PDA), and incubated at 22 ± 2°C. Only a Fusarium spp. was consistently isolated in pure culture. Twenty healthy potted night-blooming cereus plants, 10 of them previously injured with needles on the rootstock near the soil line, were gently removed from the substrate and inoculated by a 1-min immersion of their base in a suspension of 1.4 × 106 conidia per ml of sterile distilled water, prepared from 15-day-old cultures. Ten control plants, five of them previously injured, were immersed in sterile distilled water. Inoculated and noninoculated plants were replanted in the original substrate, placed in a climatic chamber at 22 ± 2°C, and monitored for disease expression. Basal rot was observed on all injured inoculated plants 12 days after inoculation. Symptoms on undamaged plants appeared 22 days after inoculation. After 72 days of incubation, all inoculated plants were totally rotten. Control plants remained symptomless. The same pathogen was reisolated to fulfill Koch's postulates. For species identification, single-spore cultures were grown on PDA and carnation leaf agar in a climatic chamber at 23 ± 2°C with a 12-h darkness/near ultraviolet light regimen. The micromorphology and culture features, mainly conidial ontogeny, were consistent with descriptions of Fusarium oxysporum Schlechtend.:Fr. (1). The pathogen was able to penetrate undamaged tissues. Needle injuries accelerated infection. To our knowledge, this is the first report of Fusarium oxysporum on H. undatus in Gran Buenos Aires, Argentina. A culture of the pathogen was deposited at the fungal collection of PRHIDEB-CONICET (University of Buenos Aires) as BAFCult 3158. Reference: (1) P. E. Nelson et al. Fusarium species. An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, PA, 1983.

First Report of Species of Colletotrichum Causing Leaf Blotch of Liriodendron tulipifera in Argentina.

Since 1986, irregular, blotchy lesions of leaves and petioles of tulip tree (Liriodendron tulipifera L.) have been observed each summer in the city of La Plata, Argentina. Leaf spots are initially 2 to 3 mm in diameter and water soaked, and then they become brown or black with faint chlorotic margins that are more noticeable on the adaxial surface. Lesions may attain a diameter of 12 mm. Veins and petioles may also be affected and lesions may coalesce, especially at leaf margins. Surface-sterilized pieces of necrotic tissue collected from eight sites in the La Plata area were plated on potato dextrose agar and incubated at 27°C. Two Colletotrichum species were consistently isolated from the lesions. Single-spore cultures were obtained and identified as C. acutatum (1) and C. gloeosporioides (4). When cultured on V8 medium, mycelial growth of C. acutatum was tufted and pale gray, and the reverse side of colonies was buff to cream or pale gray to tan, but never dark. Hyaline conidia were buff to rosy buff en masse and elliptical or elongated with abruptly tapering ends measuring 8.04 to 13.58 × 2.68 to 3 µm. Micromorphology of conidia were compared with C. acutatum "Herb IMI 232176" (1). C. gloeosporioides cultures were dark gray; their reverse was dark mouse gray to fuscous black. The cirrhus was peach to orange and hyaline conidia were oblong with rounded ends measuring 8.6 to 13.40 × 2.68 to 4.02 µm. Pathogenicity was verified on greenhouse-grown tulip trees and strawberry cv. Pájaro (Fragaria × ananassa) by spray inoculation with conidial suspensions of C. acutatum (107/ml), C. gloeosporioides (106/ml), and a mixture of both species (equal parts of each suspension). Controls were sprayed with sterile distilled water. Irregular, blotchy lesions of tulip tree appeared 30 days after inoculation. Anthracnose symptoms (3) were observed on strawberries 7 days after inoculation. In all cases, lesions produced by the mixture were larger than those obtained with individual species. The inoculated fungi were reisolated from lesions of all species tested, fulfilling Koch's postulates. No lesions were observed on control plants. Voucher specimens were deposited in La Plata Spegazzini Herbarium, Argentina as C. acutatum (LPS 47187) and C. gloeosporioides (LPS 47188); cultures of both fungi were also deposited as LPSC 795 and LPSC 796, respectively. C. acutatum and C. gloeosporioides cause Liriodendron leaf blotch in Argentina. Glomerella cingulata was previously reported affecting L. tulipifera (2), but to our knowledge, this is the first report of tulip tree as a host of C. acutatum. References: (1) B. J. Dyko and J. E. M. Mordue. No. 630 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1979 (2) K. Ito and T. Kobayashi. Bull. For. Exp. Stn. Meguro 146:1, 1962. (3) C. J. Ramallo et al. Plant Dis. 84:706, 2000. (4) J. A. von Arx. Phytopathol. Z. 29:413, 1957.

[Population of Fusarium spp. in soils from the arid regions and effect of water potential on their growth]. / Población de Fusarium spp en suelos de zonas áridas e influencia del potencial agua sobre su crecimiento.

In trials of wheat establishment in irrigated areas in the Patagonian valleys (Picún Leufú), sectors with plants showing symptoms of typical foot-rot were detected. In order to investigate the phytopathogenic mycoflora of these soils (arid zone), the population of Fusarium spp was analysed quali- and quantitatively and its tolerance to water stress by measuring the mycelial growth in the presence of different water potentials. Fusarium spp detected in that type of soil did not coincide with those commonly present in traditional wheat zones. In 100% of the samples analysed the predominant fungus present vas Fusarium equiseti. This species was the one isolated from wheat plants with foot-rot. It was isolated in soil sown with wheat as well as in soils free of wheat. In none of the samples, was the number less than 10,000 propagules/g of soil. In sporadic cases F. moniliforme and F. oxysporum appeared. When comparing, in different water potentials, the growth capacity of isolated species in the arid zone with respect to the other phytopathogenic species common in wheat (F. graminearum an F. poae), it was noted that F. equiseti showed the greatest tolerance to water stress. This species had the capacity to grow in water potentials as low as -12 MPa. This physiological characteristic was manifest in the strains of F. equiseti found in the arid zone and also in those isolated in a wet zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Población de Fusarium spp en suelos de zonas áridas e influencia del potencial agua sobre su crecimiento. / [Population of Fusarium spp. in soils from the arid regions and effect of water potential on their growth]

In trials of wheat establishment in irrigated areas in the Patagonian valleys (Picún Leufú), sectors with plants showing symptoms of typical foot-rot were detected. In order to investigate the phytopathogenic mycoflora of these soils (arid zone), the population of Fusarium spp was analysed quali- and quantitatively and its tolerance to water stress by measuring the mycelial growth in the presence of different water potentials. Fusarium spp detected in that type of soil did not coincide with those commonly present in traditional wheat zones. In 100

of the samples analysed the predominant fungus present vas Fusarium equiseti. This species was the one isolated from wheat plants with foot-rot. It was isolated in soil sown with wheat as well as in soils free of wheat. In none of the samples, was the number less than 10,000 propagules/g of soil. In sporadic cases F. moniliforme and F. oxysporum appeared. When comparing, in different water potentials, the growth capacity of isolated species in the arid zone with respect to the other phytopathogenic species common in wheat (F. graminearum an F. poae), it was noted that F. equiseti showed the greatest tolerance to water stress. This species had the capacity to grow in water potentials as low as -12 MPa. This physiological characteristic was manifest in the strains of F. equiseti found in the arid zone and also in those isolated in a wet zone.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relation between the production of deoxynivalenol and zearalenone and the mycelial growth of Fusarium graminearum on solid natural substrates]. / Relación entre la producción de deoxinivalenol y zearalenona y el crecimiento micelial de Fusarium graminearum en sustratos naturales sólidos.

A toxicogenic strain of Fusarium graminearum which produces DON and ZEA was cultivated on natural solid substrates (wheat, polished rice and hulled rice) under different environmental conditions. The production of both toxins and mycelium growth (in terms of glucosamine) were evaluated to establish the relation between the production of DON and ZEA and the different mycelium growth on the substrates mentioned above. Polished rice was the substrate on which most production of both toxins was obtained. Comparing the three substrates studied, the highest quantities of DON were obtained at a temperature of 27 degrees C during incubation period, being indifferent to the presence of light except in the case of hulled rice. Whereas for ZEA the best conditions in wheat and polished rice were medium temperatures (17 degrees-21 degrees C respectively) and darkness. While in hulled rice the ideal conditions for the production of both toxins were temperature of 27 degrees C and the presence of light. Concerning the mycelium growth, this was very scarce when cultivated in hulled rice, increasing in polished rice and being largest in wheat. The increase or reduction of the mycelium growth in the different substrates was not proportional to the increase or decrease of the production of both toxins. Therefore, production of DON and ZEA could be subjected to the nature of the substrate and environmental conditions, more than the rate of development of Fusarium graminearum in cereal grains.

[The genus Fusarium in Argentina. II. Identification of species which affect crops in the La Plata region]. / Género Fusarium en la Argentina. II. Identificación de especies que afectan cultivos en la zona de La Plata.

Isolations from different hosts were performed to identify by their morphological, biometric and culture characteristics the species of the genus Fusarium commonly affecting crops in La Plata and its surroundings. An identification index was designed with the species isolated and a comparative chart was constructed through a revision of the genus to establish equivalents between the main classification systems.

Género Fusarium en la Argentina. II. Identificación de especies que afectan cultivos en la zona de La Plata. / [The genus Fusarium in Argentina. II. Identification of species which affect crops in the La Plata region]

Isolations from different hosts were performed to identify by their morphological, biometric and culture characteristics the species of the genus Fusarium commonly affecting crops in La Plata and its surroundings. An identification index was designed with the species isolated and a comparative chart was constructed through a revision of the genus to establish equivalents between the main classification systems.