ABSTRACT
Chile leads cherry exports in the southern hemisphere with a total of 415.315 t exported in the 2022 to 2023 season (IQonsulting, 2023). Cytospora canker, produced by Cytospora spp., causes destructive infections and limit the productivity of sweet cherry orchards (Luo et al. 2019). This study was focused on isolating Cytospora strains to identify and characterize the species present in sweet cherry. During the period 2019-2022, ten samples of stem or branch presenting canker, dieback, gummosis or dead buds, were collected from sweet cherry cultivars 'Skeena', 'Lapins', 'Santina', 'Sweetheart', and 'Regina', in the regions Ñuble and O'Higgins, Chile. Five mm pieces from the necrotic wood margins of the samples were rinsed with sterile deionized water, placed on potato dextrose agar (PDA, Difco) and incubated at 20±2 ºC for 5 days. One isolate was recovered from each sample, resulting in ten Cytospora-like strains. Single hyphal tips were transferred onto PDA plates and all isolates were deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM). Colonies grown on PDA reached 89 mm in diameter in 10 d at 25 °C, showing irregular margin, lacking aerial mycelium, initially off-white to cream that turned greenish gray in the center, which darkens with age. After 20 days of culturing on pine needle agar (Chen et al. 2015), isolates produced conidiomata pycnidial, semi-immersed, black, and subglobose (362)445-555(681)×(357)528-700(1053) µm (n=10), generating amber slimy conidia masses; Conidiophores were phialidic, cylindrical, aseptate, hyaline (6.77)9-10.04(12.88)×(0.82)1.1-1.28(1.99) µm (n = 30); conidia were abundant, allantoid, hyaline to light brown, aseptate (3.39)4.28-4.57(5.36)×(0.69)0.96-1.09(1.47) µm (n = 30) (Supplementary Figure 1). No sexual morph was observed. With the exception of the strain RGM 3390, all the isolates shared morphological characters to the descriptions of Cytospora sorbicola Norphanph., Bulgakov, T. C. Wen & K. D. Hyde (Norphanphoun et al. 2017). Isolates were identified at species level, by sequencing DNA regions described by Pan et al. (2020): ITS1-5.8S-ITS2, LSU; act, tef-1α, and tub2 with the exception of the RBP2, because this region could not be amplified in seven out of ten isolates. The consensus tree included the concatenated sequences of the ten isolates and those of reference Cytospora species reported by Ilyukhin et al. (2023) using a maximum likelihood analysis with the tool IQ-TREE webserver. MLSA confirmed the taxonomic affiliation of nine of the isolates with C. sorbicola and one isolate with Cytospora sp. (RGM 3390), that might represent a novel species (Supplementary Figure 2). The isolates RGM 3399 and RGM 3400, were selected randomly for pathogenicity tests. Inoculations were performed on 2-year-old sweet cherry cv. 'Lapins' grow in pots in a greenhouse at 26±6°C. Seven plants per isolate were cut to about 6-cm length from the main stem, and inoculated onto fresh cuts with 5-mm mycelium PDA plugs of 5-d-old culture and wrapped in moist sterile cotton and parafilm to keep moisture. Six plants were inoculated with non-colonized PDA agar plugs as control. The average canker length 3 months after inoculation was 3.1 and 0.8 cm, for RGM 3389 and RGM 3400, respectively (Supplementary Figure 1). Symptomatic twigs were incubated in moist chambers at 20±2 ºC for 10 d, resulting in the re-isolation of Cytospora strains that produced pycnidia and conidia structures in agreement with C. sorbicola. Both strains were reidentified to fulfill Koch's postulates, control twigs remained asymptomatic and no fungus was isolated from these twigs. This is the first report of C. sorbicola causing canker on sweet cherry in Chile. Our findings suggest that this species could be the most recurrent in cherry in central Chile, coinciding with it found in California where C. sorbicola has been described as the main causal agent of Cytospora canker of stone fruits in California (Lawrence et al. 2018).
ABSTRACT
Gummosis is an aggressive disease caused by the necrotrophic fungus Lasiodiplodia theobromae (Pat.) Griffon & Maubl that threatens commercial cashew orchads in Brazil. To study the molecular mechanisms involved in the cashew response to L. theobromae, a proteomic analysis of stems from the commercial cashew clone BRS 226 (resistant) was conducted at early times post-artificial infection. In addition, changes in the stem proteome profiles of gummosis resistant and susceptible cashew plants grown under field condition and naturally exposed to pathogen were also compared. After two-dimensional gel electrophoresis (2D-PAGE), 73 proteins showed statistically significant differences in spot abundance. Of these, 31 spots were identified in BRS 226 stems compared with mock-inoculated controls and 32 in stems collected from field-grown resistant and susceptible cashew plants. L. theobromae-responsive proteins were mainly involved in energy metabolism pathways, stress and defense, cell signaling and protein metabolism indicating modulation of various cellular functions upon fungal infection. As stress-inducing factors seem to be important for susceptibility to disease, the change in the abundance relative these proteins may possibly indicate an attempt to maintain cellular homeostasis, as resistance determinant factor, related with a possible role in the regulation of oxidative burst. These findings provide the first information about the cellular mechanisms acting in the Anacardium occidentale genotypes associated with the pathophysiological state of infection with L. theobromae. BIOLOGICAL SIGNIFICANCE: Gummosis caused by Lasiodiplodia theobromae, a necrotrophic fungus, is the major disease of cashew plants in the semi-arid conditions of northeastern Brazil. Although various studies were carried out on this pathosystem, there is no information available on the molecular mechanisms of plant defense related to the incompatible interaction of cashew with L. theobromae. Therefore, this original study comprises a differential proteomic analysis of cashew stems from: (i) resistant dwarf clone BRS 226 mock-inoculated (control) and artificially inoculated with L. theobromae; and (ii) cashew plants bearing resistant and susceptible traits to gummosis, originated from open pollination of BRS 226 in a commercial orchard with high disease incidence. The contribution of the reprogrammed proteins to molecular events triggered in cashew plants challenged by L. theobromae has a great relevance in the identification of the host candidate proteins linked to biological pathways that respond to L. theobromae infection. Furthermore this study may contribute to improve breeding programs aimed at selecting resistant/tolerant cashew clones toward this pathogen.
Subject(s)
Anacardium/metabolism , Ascomycota , Disease Resistance/physiology , Plant Diseases/microbiology , Plant Proteins/biosynthesis , Proteomics , Anacardium/microbiologyABSTRACT
Gummosis is among the main fungal diseases of the citrus. It is caused by Phytophthora sp. and usually shows up in the lap of the plant, provoking rottenness and gum exudation, and expands causing the plant death for constrictions in the cambium or phloem which interrupts the descending fow of sap. The objective of this work was to evaluate the antagonistic in vitro activity of Trichoderma spp. to the fungi Phytophthora citrophthora. Phytophthora citrophthora was exposed to fve environments of antagonism (without antagonist and with four strains of Trichoderma viride, T. virens, T. harzianu and T. stromaticum). The in vitro essay was accomplished through the method of paired cultures. A completely randomized desing was used with fve treatments and three replications, and each plot was represented by three petri dishes. The isolates of Trichoderma demonstrated signifcant effect in the inhibition of the mycelial growth of the fungi Phytophthora citrophthora, and the fungi Trichoderma stromaticum presented larger antagonism to the fungi P. citrophthora while the T. harzianum presented antagonism smaller.
A citricultura nacional apresenta inúmeras pragas e doenças que limitam sua produção dentre as principais doenças fúngicas da cultura dos citros pode se destacar a gomose. A gomose causada por Phytophthora sp. geralmente se manifesta no colo da planta, provocando podridão e exsudação de goma, podendo expandir-se ocasionando a morte da planta por estrangulamento devido ao ataque do cambio ou foema, o que interrompe o fuxo descendente de seiva. Este trabalho foi realizado com o objetivo de se avaliar a atividade antagônica in vitro, de isolados de Trichoderma spp. ao fungo Phytophthora citrophthora. O fator em estudo foi um patógeno (Phytophthora citrophthora) em cinco ambientes de antagonismo (sem antagonista e com os isolados de Trichoderma viride, T. virens, T. harzianu e T. stromaticum). O ensaio in vitro foi realizado por meio do método de cultura pareada. O delineamento experimental utilizado foi inteiramente casualizado, com 5 tratamentos e 3 repetições, e cada parcela foi representada por três placas de Petri. Os isolados de Trichoderma demonstraram efeito signifcativo na inibição do crescimento micelial do fungo Phytophthora citrophthora, sendo que o fungo Trichoderma stromaticum apresentou maior antagonismo ao fungo P. citrophthora enquanto que o T. harzianum apresentou menor antagonismo.
ABSTRACT
Gummosis is among the main fungal diseases of the citrus. It is caused by Phytophthora sp. and usually shows up in the lap of the plant, provoking rottenness and gum exudation, and expands causing the plant death for constrictions in the cambium or phloem which interrupts the descending fow of sap. The objective of this work was to evaluate the antagonistic in vitro activity of Trichoderma spp. to the fungi Phytophthora citrophthora. Phytophthora citrophthora was exposed to fve environments of antagonism (without antagonist and with four strains of Trichoderma viride, T. virens, T. harzianu and T. stromaticum). The in vitro essay was accomplished through the method of paired cultures. A completely randomized desing was used with fve treatments and three replications, and each plot was represented by three petri dishes. The isolates of Trichoderma demonstrated signifcant effect in the inhibition of the mycelial growth of the fungi Phytophthora citrophthora, and the fungi Trichoderma stromaticum presented larger antagonism to the fungi P. citrophthora while the T. harzianum presented antagonism smaller.
A citricultura nacional apresenta inúmeras pragas e doenças que limitam sua produção dentre as principais doenças fúngicas da cultura dos citros pode se destacar a gomose. A gomose causada por Phytophthora sp. geralmente se manifesta no colo da planta, provocando podridão e exsudação de goma, podendo expandir-se ocasionando a morte da planta por estrangulamento devido ao ataque do cambio ou foema, o que interrompe o fuxo descendente de seiva. Este trabalho foi realizado com o objetivo de se avaliar a atividade antagônica in vitro, de isolados de Trichoderma spp. ao fungo Phytophthora citrophthora. O fator em estudo foi um patógeno (Phytophthora citrophthora) em cinco ambientes de antagonismo (sem antagonista e com os isolados de Trichoderma viride, T. virens, T. harzianu e T. stromaticum). O ensaio in vitro foi realizado por meio do método de cultura pareada. O delineamento experimental utilizado foi inteiramente casualizado, com 5 tratamentos e 3 repetições, e cada parcela foi representada por três placas de Petri. Os isolados de Trichoderma demonstraram efeito signifcativo na inibição do crescimento micelial do fungo Phytophthora citrophthora, sendo que o fungo Trichoderma stromaticum apresentou maior antagonismo ao fungo P. citrophthora enquanto que o T. harzianum apresentou menor antagonismo.
ABSTRACT
The behavior of the mandarin 'Ponkan', tangor 'Murcot' and orange 'Valência', artificially inoculated with two isolates of the Dothiorella gregaria, was evaluated under different seasons of the year an under nutritional and phenological states. During the period between december and november, in a subsequent phase of harvesting of the fruits, the plants had showed more affected by the isolated fungus. 'Ponkan' revealed to be more affected, followed by 'Murcot' and 'Valência'. The suscetibility of the species showed more severity of the disease under favorable conditions. The plants showing lower contents of Ca and Zn reached the highest rates of lesion, and they were in the subsequent phase of harvesting. The results indicated the effect of the inoculation time in the severity of gomosis caused by D. gregaria and confirm the predisposition of the weakened plants nutritionaly.
O comportamento da tangerineira "Ponkan", da tangoreira "Murcot" e da laranjeira "Valência" inoculadas artificialmente com dois isolados de Dothiorella gregaria foi avaliado sob diferentes épocas do ano, estados nutricionais e fenológicos. No período compreendido entre os meses de novembro a dezembro, em fase posterior à colheita dos frutos, as plantas mostraram-se mais afetadas pelos isolados do fungo. "Ponkan" mostrou-se mais afetada, seguida por "Murcot" e "Valência", observando-se a mesma suscetibilidade das espécies nos diferentes períodos, porém com maior severidade da doença sob condições de elevada precipitação e umidade relativa. As plantas que apresentaram menores teores de Ca e Zn atingiram maiores índices de lesão, ocasião em que se encontravam em fase posterior à colheita. Os resultados indicam o efeito da época de inoculação na severidade da gomose causada por D. gregaria e confirmam a predisposição de plantas mais debilitadas nutricionalmente.
ABSTRACT
The behavior of the mandarin 'Ponkan', tangor 'Murcot' and orange 'Valência', artificially inoculated with two isolates of the Dothiorella gregaria, was evaluated under different seasons of the year an under nutritional and phenological states. During the period between december and november, in a subsequent phase of harvesting of the fruits, the plants had showed more affected by the isolated fungus. 'Ponkan' revealed to be more affected, followed by 'Murcot' and 'Valência'. The suscetibility of the species showed more severity of the disease under favorable conditions. The plants showing lower contents of Ca and Zn reached the highest rates of lesion, and they were in the subsequent phase of harvesting. The results indicated the effect of the inoculation time in the severity of gomosis caused by D. gregaria and confirm the predisposition of the weakened plants nutritionaly.
O comportamento da tangerineira "Ponkan", da tangoreira "Murcot" e da laranjeira "Valência" inoculadas artificialmente com dois isolados de Dothiorella gregaria foi avaliado sob diferentes épocas do ano, estados nutricionais e fenológicos. No período compreendido entre os meses de novembro a dezembro, em fase posterior à colheita dos frutos, as plantas mostraram-se mais afetadas pelos isolados do fungo. "Ponkan" mostrou-se mais afetada, seguida por "Murcot" e "Valência", observando-se a mesma suscetibilidade das espécies nos diferentes períodos, porém com maior severidade da doença sob condições de elevada precipitação e umidade relativa. As plantas que apresentaram menores teores de Ca e Zn atingiram maiores índices de lesão, ocasião em que se encontravam em fase posterior à colheita. Os resultados indicam o efeito da época de inoculação na severidade da gomose causada por D. gregaria e confirmam a predisposição de plantas mais debilitadas nutricionalmente.