Laboratorio de Patología Frutal, Universidad de Talca, Chile

Methods to Stimulate Sporulation and Freeze-Drying Strategies for the Conservation of Diplodia mutila, Diplodia seriata, Lasiodiplodia theobromae, and Neofusicoccum arbuti Isolated from Apple Trees with Canker and Dieback Symptoms

Abstract

Four Botryosphaeria spp. isolated from apple with dieback symptoms, Diplodia mutila, Dip-lodia seriata, Neofusicoccum arbuti, and Lasiodiplodia theobromae, were subjected to different conditions to induce sporulation, then freeze-dried and stored in glass vials and ampoules at a temperature of 4 °C using two protective media (skimmed milk powder in water at 20% and a mixture of skimmed milk powder at 20% plus 5% inositol). Viability was as-sessed after storage periods of 1, 90, 180, and 365 days. Low-nutrient growth conditions on 2% water agar supplemented with pine needles, incubated under UV light (λ = 350 nm) and at 24 °C, effectively stimulated sporulation of all four Botryosphaeriaceae species. The survival rate of the isolates was similar. Overall viability showed slight but significant differences depending on the type of protective medium and storage container used for the freeze-dried cultures (p < 0.001). Among the tested media, the highest viability was maintained in vacuum-sealed glass ampoules using either a medium containing 20% skimmed milk powder with 5% inositol or 20% skimmed milk powder alone.

Published on line: 29 August 2025

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https://www.mdpi.com/2309-608X/11/9/640

Efficacy of pruning-wound protectant fungicides for the management of Botryosphaeria canker and dieback of apple trees in Maule region, Chile

Published Online:25 Mar 2025
https://doi.org/10.1094/PDIS-11-24-2427-RE

Abstract
Botryosphaeria canker and dieback is an important fungal disease caused by Botryosphaeriaceae spp. that affects the productivity of apple orchards in Chile and worldwide. In the field, studies on the management of this disease are focused on the protection by fungicides applied on pruning wounds. However, in Chile, information about the protection and efficacy of fungicides on apple trees against twig and branch pathogens is scarce. Therefore, in this study, we evaluated the efficacy of fungicides to control fungal trunk pathogens causing Botryosphaeria canker and dieback in apple trees using in vitro, glasshouse, and field trials. Isolates of Diplodia mutila, D. seriata, Neofusicoccum arbuti, and Lasiodiplodia theobromae obtained and characterized previously in Chile from apple trees with canker and dieback symptoms were used in this study. In vitro tests showed that benomyl, fluazinam, difenoconazole, and tebuconazole exhibited the lowest EC50 values with means of 0.08, 0.09, 0.12, and 0.18 μg/mL, respectively. This study demonstrates that infection caused by D. mutila, D. seriata, L. theobromae, and N. arbuti can be significantly reduced using single-sprayed protection of fungicides. The most effective in reducing infection on pruning wounds of apple trees by Botryosphaeriaceae were benomyl (66 to 76%), tebuconazole (47 to 68%), thiophanate-methyl (68 to 71%), boscalid + pyraclostrobin (47 to 63%), fluxapyroxad + pyraclostrobin (54 to 57%), and thiophanate-methyl + tetraconazole (63 to 74%). To our knowledge, this is the first study reporting the control of Botryosphaeriaceae causing canker and dieback in apple trees through the use of commercially available chemical fungicides, with different active ingredients and modes of action.

https://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-11-24-2427-RE

Abstract

In recent years, the number of apple trees affected by Botryosphaeria cankers and dieback has considerably increased in central Chile. This study aimed to identify the species of Botryosphaeriaceae associated with canker and dieback symptoms, estimate disease incidence and distributions, and study their pathogenicity and virulence on apple and other fruit crops. A field survey of 34 commercial orchards of apple (7 to 30 years old) was conducted in 16 localities, obtaining 270 symptomatic branch and trunk samples in 2017 and 2018 growing seasons. The incidence of Botryosphaeria canker and dieback ranged between 5 and 40%, and a total of 255 isolates of Botryosphaeriaceae spp. were obtained from 238 cankers. Morphological identification along with phylogenetic studies of the internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) of the rDNA, part of the translation elongation factor 1-α (tef1-α), and part of the β-tubulin (tub2) genes allowed us to identify Diplodia mutila (n = 49 isolates), D. seriata (n = 136 isolates), Lasiodiplodia theobromae (n = 16 isolates), and Neofusicoccum arbuti (n = 54 isolates). L. theobromae was isolated mainly from apple dieback from northern localities. All pathogens tested were pathogenic, causing canker and dieback symptoms on lignified twigs of apple, pear, walnut, and green grapevine shoots in the field. Isolates of N. arbuti were the most virulent, reproducing more severe cankers on the lignified tissues inoculated. This study reports, for the first time, D. mutila and L. theobromae associated with Botryosphaeria canker and dieback in Chile, and it is the first description of N. arbuti causing apple dieback worldwide.

https://apsjournals.apsnet.org/doi/full/10.1094/PDIS-06-21-1291-RE

Abstract

Studies on susceptibility of the age of the pruning wound and effect of pruning time on infection by Botryosphaeriaceae in apple trees is scarce. This work aimed to determine the susceptibility of different ages of pruning wound (1, 15, 30 or 45 days after pruning) and the effect of pruning time during early (June) and late pruning (August) in apples cvs. Fuji and Gala on the infection of Diplodia mutila, D. seriata, Neofusicoccum arbuti and Lasiodiplodia theobromae. Additionally, seasonal conidial release of Botryosphaeriaceae spp. was monitored using Vaseline-coated glass slides in two orchard cv. Fuji and Gala during two consecutive growing seasons (2020-2021) in Maule region, Central Chile. The analysis of the variance of each pruning wound age showed significant interaction between the factors, Botryosphaeria isolate × apple cultivar (P= 0.0001). The isolates of N. arbuti and D. seriata were significantly more aggressive than the other isolates of Botryosphaeria, with mean values of 45.2 mm (one day of age) to 7.9 mm (45 days of age) and 31.0 mm (one day of age) to 6.6 mm (45 days of age), respectively. Independently of the Botryosphaeriaceae species, the pruning wound susceptibility considerably decreased as the age of the pruning wound increased, where fresh pruning wounds (1 and 15 days) are more susceptible than older pruning wounds (30 and 45 days). Our results indicated significant (P < 0.0001) interaction between Botryosphaeria isolates and pruning time for both cultivars. Necrotic lesions varied among the Botryosphaeriaceae species, where lesion length was significantly greater in the isolates of N. arbuti, D. mutila, and D. seriata inoculated in early pruning than inoculations conducted in late pruning. This study demonstrated a strong relationship between conidial release and rainfall, where 70.3% and 86.3% of the total conidia of Botryosphaeriaceae spp. were released during rainfall periods between June and August (winter). This work suggests that a delay in pruning times may be justified to reduce the risk of Botryosphaeria canker and dieback in apple trees in Maule region, Chile, considering rain-free days.

https://apsjournals.apsnet.org/doi/abs/10.1094/PDIS-07-24-1498-RE?journalCode=pdis

Reacción en cadena de la polimerasa (PCR)‼️

Es un procedimiento que permite obtener grandes cantidades de segmentos específicos de ADN, es decir, se trata de una replicación acelerada in vitro de segmentos de ADN. Consiste en colocar en un mismo espacio todos los elementos necesarios para la síntesis de ADN: ADN molde, cebadores, ADN polimerasa y desoxirribonucleótidos libres. Todos ellos se encuentran en un tubo de ensayo colocado dentro de un termociclador, que es capaz de proporcionar grandes variaciones de temperatura en pequeños periodos de tiempo, consiguiendo así tres pasos fundamentales:
(a) Desnaturalización del ADN molde: se consigue a una temperatura de 90–95 °C. Esto permite la ruptura de los enlaces de hidrógeno que unen ambas hebras de ADN, dejando expuestas las bases nitrogenadas.
(b) Unión de los cebadores (2) por complementariedad al ADN molde: se consigue disminuyendo la temperatura a 35–52 °C.
(c) Elongación de la cadena de ADN: se obtiene a 70–72 °C. La Taq polimerasa colocará los nucleótidos según lo que lea sobre el ADN molde.

La polimerasa debe ser termorresistente; por ello, la ADN polimerasa utilizada proviene de una bacteria cuyo hábitat natural es el agua de unos géiseres: Thermus aquaticus. Esta polimerasa se llama Taq (T de Thermus, aq de aquaticus) polimerasa.

En el primer ciclo, a partir de una copia del gen de interés (representado en color verde), se obtendrán dos copias. Tras 40 ciclos, se habrán producido millones de copias.

El Ciclo Celular en los Organismos Procarióticos‼️

Una célula recién formada de un organismo procariótico, como la bacteria Escherichia coli, debe aumentar de tamaño, replicar su cromosoma circular y luego mover cada uno de los dos cromosomas hijos resultantes a su propia célula progenie durante la división celular. Todo el mecanismo de división de las células procarióticas, llamado fisión binaria (división o división en dos partes), se puede considerar en tres períodos, como se muestra en la figura. Una vez que se forman las células hijas, pueden crecer durante algún tiempo (período B) antes de iniciar la síntesis de ADN. Una vez que los cromosomas se replican y se separan en extremos opuestos de la célula (período C), la membrana se pellizca entre ellos y se forman dos células hijas (período D).

El ciclo celular bacteriano. Durante el período B, desde el nacimiento hasta el inicio de la replicación del ADN, la célula aumenta de tamaño. El cromosoma se replica y los cromosomas hijos resultantes se mueven a extremos opuestos durante el período C. Luego, la célula se divide por fisión binaria durante el período D. En cultivos de muy rápido crecimiento, el período B puede no existir; ¡Las células hijas pueden formarse con cromosomas que ya están parcialmente replicados!

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