Effect of Trichoderma on horticultural seedlings' growth promotion depending on inoculum and substrate type.

AIMS: The biostimulant effect of Trichoderma spp. on horticultural crops are highly variable. Thus, practical use of Trichoderma sp. requires feasible formulated products and suitable substrates. METHODS AND RESULTS: This study evaluates the survival and the growth-promotion effect of a Trichoderma saturnisporum rice formulation compared with a nonformulated conidia suspension (seven treatments in total), on tomato, pepper and cucumber seedlings grown in two substrates: (i) rich in organic matter (OM) and (ii) mineral substrate without OM. The results showed beneficial effects on seedling growth in the OM-rich substrate when T. saturnisporum rice formulation (mainly at maximum concentration) was applied, but the effects were opposite when the mineral substrate without OM was used. The effects were closely linked to the level of inoculum in the substrate, which was greater upon application of the formulated inoculum as opposed to the nonformulated one. CONCLUSIONS: The use of rice to prepare the inoculum of T. saturnisporum seems to be promising for seedling growth in the nursery when it is applied in a substrate that is rich in organic matter, but it must be considered that under certain conditions of food shortage, Trichoderma sp. could show pathogenicity to seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence of the complexity inherent in the use of micro-organisms in agriculture, while also confirming that the activity of the biofertilizers based on Trichoderma depends on the type of inoculum and its concentration, as well as the properties of the medium in which the fungi develop. Further studies assessing the effectiveness or possible pathogenicity of Trichoderma in different soils under greenhouse conditions must be addressed.

First Report of Fusarium oxysporum on Sweet Pepper Seedlings in Almería, Spain.

In March of 2013, new symptoms were observed in more than seven million nursery-grown sweet pepper (Capsicum annuum) plants in El Ejido, Almería (southern Spain). Symptoms included wilting without yellowing of leaves and stunting of plants. Plant crowns exhibited necrosis that advanced through the main root along with slight root rot. Xylem was not affected above or below the crown. Symptoms were thought to be caused by the well-known pepper pathogen Phytophthora capsici. However, sporodochia of Fusarium oxysporum were observed on plant crowns. Symptomatic seedlings (n = 200) were sampled and analyzed. Tissue from roots and epidermal crowns were plated on PDA, PARP, and Komada media, as well as stem discs on PDA and Komada. No Phytophthora sp. were observed and F. oxyporum was exclusively isolated from all 200 samples, from roots and crowns, but not from xylem. Pathogenicity of 60 of these F. oxysporum isolates was studied by inoculation onto sweet pepper plants (cv. del Piquillo) at the 2-true-leaf stage. Twelve plants per isolate, grown on autoclaved vermiculite, were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate per plant. Each suspension was obtained by blending one PDA petri dish fully covered with one isolate. Non-inoculated plants served as control. Plants were maintained for 30 days in a growth chamber with a 14-h photoperiod (1.6 ×·104 lux) and temperatures at 23 to 26°C. The assay was conducted twice. Symptoms described above were reproduced on crown and roots of the inoculated plants with no symptoms in stem discs. No symptoms were observed on controls after 48 days. Host specificity was tested for 13 isolates to tomato (Solanum lycopersicum) cv. San Pedro, eggplant (S. melongena) cv. Alegria, cucumber (Cucumis sativus) cv. Marketmore, watermelon (Citrullus lanatus) cv. Sugar Baby, and Chinese cabbage (Brassica campestris subsp. condensa) cv. Kasumi (4). These plants were inoculated as previously described for pathogenicity tests (12 plants per species, repeated twice). None of the plants exhibited the characteristic symptoms after 60 days. Five isolates of F. oxysporum f. sp. radicis-cucumerinum and four isolates of F. o. f. sp radicis-lycopersici were also inoculated without any symptoms in any of the inoculated sweet pepper plants. Morphological identity of all isolates corresponded to F. oxysporum. The fungi were identified following the morphological keys and methodology provided by (1) and (2). Three isolates from the 60 tested were selected for molecular identification. Molecular identification was performed by sequencing partial TEF-1α gene (3). Subsequent database searches by BLASTn indicated that the resulting sequence of 659-bp had 100% identity with the corresponding gene sequence of F. oxysporum. The sequences were identical for the three isolates and were deposited on the EMBL Sequence Database (HG916993, HG916994, and HG916995). Results suggest that the pathogenic ability of the isolates varies from a vascular Fusarium wilt. F. oxysporum f. sp. capsici is a reported pathogen to sweet pepper (5), but the symptoms we have found are closer to those manifested by the formae speciales that causes root and crown rot of other plants. Consistent with the convention stablished for similar diseases we propose the name F. oxysporum f. sp. radicis-capsici f. sp. nov. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (2) P. E. Nelson et al. Fusarium species. An Ilustrated Manual for Identification. The Penn St. University Press, 1983. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. 95:2044, 1998.(4) L. M. Oelke and P. W. Bosland. Capsicum Eggplant Newsl. 20:86, 2001. (5) V. C. Rivelli. M.S. Thesis. Dep. Plant Pathol. and Crop Phys. Louisiana State Univ., Baton Rouge, 1989.

Evaluation of alternatives to methyl bromide in melon crops in Guatemala.

The monoculture of melon in Guatemala has caused the massive appearance of plants with an analogous syndrome for the well-known disease commonly called melon collapse, or vine decline, causing significant losses in crops. Methyl bromide is commonly used to sterilize soil prior to planting in Guatemala, but it must be phased out by 2015. The objective of this study was to evaluate the technique of grafting melon onto hybrids of Cucurbita (Cucurbita maxima x Cucurbita moschata), as an alternative to using soil disinfectants (such as Metam sodium, 1,3-dichloropropene, and methyl bromide) for the control of collapse. The results suggested that both soil disinfection and grafting were not necessary in these locations, since there were no statistical differences in terms of yields between the treatments and the untreated control. Furthermore, these results demonstrate that decisions to disinfect the soil must be based on the firm identification of the causal agents, in addition to preliminary assessments of yield losses.

Effects of water potential on spore germination and viability of Fusarium species.

Germination of macroconidia and/or microconidia of 24 strains of Fusarium solani, F. chlamydosporum, F. culmorum, F. equiseti, F. verticillioides, F. sambucinum, F. oxysporum and F. proliferatum isolated from fluvial channels and sea beds of the south-eastern coast of Spain, and three control strains (F. oxysporum isolated from affected cultures) was studied in distilled water in response to a range of water potentials adjusted with NaCl. (0, -13.79, -41.79, -70.37, -99.56 and -144.54 bars). The viability (UFC/ml) of suspensions was also tested in three time periods (0, 24 and 48 h). Conidia always germinated in distilled water. The pattern of conidial germination observed of F. verticilloides, F. oxysporum, F. proliferatum, F. chlamydosporum and F. culmorum was similar. A great diminution of spore germination was found in -13.79 bars solutions. Spore germination percentage for F. solani isolates was maximal at 48 h and -13.79 bars with 21.33% spore germination, 16% higher than germination in distilled water. F. equiseti shows the maximum germination percentage in -144.54 bars solution in 24 h time with 12.36% germination. This results did not agree with those obtained in the viability test were maximum germination was found in distilled water. The viability analysis showed the great capacity of F. verticilloides strains to form viable colonies, even in such extreme conditions as -144.54 bars after 24 h F. proliferatum colony formation was prevented in the range of -70.37 bars. These results show the clear affectation of water potential to conidia germination of Fusaria. The ability of certain species of Fusarium to develop a saprophytic life in the salt water of the Mediterranean Sea could be certain. Successful germination, even under high salty media conditions, suggests that Fusarium spp. could have a competitive advantage over other soil fungi in crops irrigated with saline water. In the specific case of F. solani, water potential of -13.79 bars affected germination positively. It could indicate that F. solani has an special physiological mechanism of survival in low water potential environments.

The interactive effects of temperature and osmotic potential on the growth of marine isolates of Fusarium solani.

The mycelial growth of 18 Fusarium solani strains isolated from sea beds of the south-eastern coast of Spain was tested on potato-dextrose-agar adjusted to different osmotic potentials with either KCl or NaCl (-1.50 to -144.54 bars) in 10 degrees C intervals ranging from 15 to 35 degrees C. Fungal growth was determined by measuring colony diameter after 4 days of incubation. Mycelial growth was maximal at 25 degrees C. The quantity and frequency pattern of mycelial growth of F. solani differ significantly at 15 and 25 degrees C, with maximal growth occurring at the highest water potential tested (-1.50 bars); and at 35 degrees C, with a maximal mycelial growth at -13.79 bars. The effect of water potential was independent of salt composition. The general growth pattern of F. solani showed declining growth at potentials below -41.79 bars. Fungal growth at 35 degrees C was always higher than that grow at 15 degrees C, of all the water potentials tested. Significant differences observed in the response of mycelia to water potential and temperature as main and interactive effects. The viability of cultures was increasingly inhibited as the water potential dropped, but some growth was still observed at -99.56 bars. These findings could indicate that marine strains of F. solani have a physiological mechanism that permits survival in environments with low water potential. The observed differences in viability and the magnitude of growth could indicate that the biological factors governing potential and actual growth are affected by osmotic potential in different ways.

Association of Pythium aphanidermatum with Root and Crown Rot of Melons in Honduras.

Approximately 10,000 ha of melon (Cucumis melo L.), primarily cantaloupe and honeydew types, are grown in Honduras for export to U.S. markets. In 2004 and 2005, several soil surveys were conducted in areas with a history of vine decline. Twenty-nine soil samples from six farms were collected from the rhizosphere of wilted plants. Thirty-six melon plants were planted in a mixture of each rhizosphere sample and vermiculite (1:6 v/v). The plants were maintained in a growth chamber at 23 to 25°C with a 16-h photoperiod. The first symptoms, which appeared at the one- or two-true-leaf stage, were girdling of the lower stem, leaf chlorosis, and wilting. Affected plants exhibited necrotic crowns and roots and half of all plants died less than 3 days after wilting. Isolations from washed and dried crown and roots pieces from affected plants were placed on malt extract agar. Colonies were transferred to potato carrot agar and into dishes of sterile water and immature carnation petals to aid in the identification of recovered fungi. Nearly 500 isolates of Pythium species were cultured, and approximately 60% were identified as P. aphanidermatum (Edson) Fitzp. on the basis of their toruloid sporangia, aplerotic oospores, terminal and smooth oogonia, monoclinous sac-shaped antheridia (one to two per oogonium), and abundant appressoria. The pathogenicity of nine isolates was confirmed in a growth chamber. Ten plants of melon cv. Amarillo Canario, grown in sterilized vermiculite, were inoculated at the two- or three-true-leaf stage by drenching pots with 100 ml of a suspension of each isolate (103 CFU ml-1). Noninoculated plants served as controls. There were three replicates per isolate. Plants began to die 7 days after inoculation and the incidence of the affected plants reached an average of 70%. P. aphanidermatum causing decline of melon plants has been previously reported in hot and semi-arid areas in Israel and Spain (1,2). To our knowledge, this is the first report of P. aphanidermatum pathogenic to melon plants in Honduras. References: (1) S. Pivonia et al. Plant Dis. 81:1264, 1997. (2) J. Gómez Enfermedades del Melón en los Cultivos "Sin Suelo" de la Provincia de Almería. Junta de Andalucía, 1993.

Detection of Fusarium oxysporum f. sp. melonis Race 1 in Soil in Colima State, Mexico.

During the winters of 2002 and 2003, a wilt occurred in melons cultivated on 1,500 ha in Colima State, Mexico. Yield losses reached 25% of final production, despite soil disinfestation with 60% methyl bromide and 40% chloropicrin. On the basis of the observation of plants with necrotic xylem, yellowing, and wilting of leaves, this disease was identified provisionally as Fusarium wilt. During February 2003, four soil samples from affected fields were plated onto a Fusarium-selective medium (1), which resulted in the detection of 2,260 ± 357, 179 ± 76, 668 ± 357, and 1,391 ± 256 CFU/g of F. oxysporum (3). Thirty-one randomly chosen isolates were used to inoculate differential cultivars of melon as described by Risser et al. (4). The cultivars were Amarillo Canario (susceptible to all races), Diana (resistant to races 0 and 2), Tango (resistant to races 0 and 1), and Vulcano (resistant to races 0, 1, and 2) (2). Ten plants of each cultivar, grown on sterilized vermiculite, were inoculated at the first true-leaf stage by drenching with 200 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate. Noninoculated plants of each cultivar served as controls. Plants were maintained in a growth chamber with a 16-h photoperiod (18 × 103 lux) and temperatures at 23 to 25°C. Yellowing, wilt, and vascular discoloration symptoms developed on cvs. Amarillo Canario and Diana following inoculation with each of the 31 isolates, while noninoculated plants remained symptomless. F. oxysporum was consistently reisolated on potato dextrose agar from the affected plants. On the basis of the combination of affected cultivars, all isolates were identified as F. oxysporum f. sp. melonis race 1. To our knowledge, this is the first report of F. oxysporum f. sp. melonis race 1 in Colima State, Mexico. References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) J. Marín Rodríquez. Portagrano 2004. Vadmecum de Variedades Hortícolas. Agrobook, Spain. 2004. (3) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983. (4) G. Risser et al. Phytopathology 66:1105, 1976.