Chilean Botrytis cinerea Isolates with Reduced Sensitivity to Fludioxonil Exhibit Low to Null Fitness Penalties.

The main phytosanitary problem for table grape production in Chile is gray mold caused by the fungus Botrytis cinerea. To manage this issue, the primary method utilized is chemical control. Fludioxonil, a phenylpyrrole, is highly effective in controlling B. cinerea and other plant pathogens. Consistently, there have been no field reports of reduced efficacy of fludioxonil; however, subpopulations with reduced sensitivity to fludioxonil are on the rise globally, as per increasing reports. Our study involved a large-scale evaluation of B. cinerea's sensitivity to fludioxonil in the Central Valley of Chile's primary table grape production area during the growing seasons from 2015 to 2018. Out of 2,207 isolates, only 1.04% of the isolates (n = 23) exceeded the sensitivity threshold value of 1 µg/ml. Remarkably, 95.7% are concentrated in a geographic region (Valparaíso Region). Isolates with reduced sensitivity to fludioxonil showed growth comparable with sensitive isolates and even more robust growth under nutritional deficit, temperature, or osmotic stress, suggesting greater environmental adaptation. When table grape detached berries were stored at 0°C, isolates less sensitive to fludioxonil caused larger lesions than sensitive isolates (2.82 mm compared with 1.48 mm). However, the lesions generated by both types of isolates were equivalent at room temperature. This study found no cross-resistance between fludioxonil and fenhexamid, an essential fungicide integrated with fludioxonil in Chilean B. cinerea control programs. All the Chilean isolates with reduced sensitivity to fludioxonil were controlled by the fludioxonil/cyprodinil mixture, a commonly employed form of fludioxonil. The cyprodinil sensitivity in the isolates with reduced sensitivity to fludioxonil explains their low field frequency despite their null fitness penalties. However, the emergence of fludioxonil-resistant isolates inside the Chilean B. cinerea population demands a comprehensive analysis of their genetic bases, accompanied by monitoring tools that allow the permanence of field fludioxonil efficacy.

Control of Botrytis cinerea from Chilean grapevines by pydiflumetofen: baseline and carboxamide-mutant sensitivity.

The gray mold (Botrytis cinerea; Botrytis) is the main disease affecting grapevines production in Chile. Succinate Dehydrogenase Inhibitors (SDHI) belonging to the carboxamides fungicide family are a key tool for the control of Botrytis in grapevines from Chilean Central Valley. This study aimed to determine the sensitivity of Chilean Botrytis population to the new generation carboxamide pydiflumetofen. Conidial germination (CG) and germ-tube elongation (GTE) sensitivity assays were conducted on 200 single-spore isolates collected during the 2016-2017 season. The mean effective concentration that inhibited 50% (EC50) of CG in the Botrytis population was 0.0545 µg/mL, with mean values of 0.066 µg/mL and 0.042 µg/mL, for table and wine grapes, respectively. The mean EC50 value of GTE was 0.000245 µg/mL, 0.0003 µg/mL, and 0.0019 µg/mL for the total, table grape, and wine grape populations, respectively. The comparison between pydiflumetofen and fludioxonil, a highly-efficient fungicide carrying a different mode of action, showed the 87.5% and 97.5% of Botrytis control with an EC50 threshold of 0.1 µg/mL, in table grape, and wine grape populations, respectively. No cross-resistance between pydiflumetofen and fludioxonil was detected. For nine isolates with reduced pydiflumetofen sensitivity, we evaluated SdhB mutations using a qPCR-HRM diagnostic system. Two isolates carried the sdhBP225/H272R genotype and two the sdhBP225/H272Y. Additional analysis of SdhB mutant isolates determined that pydiflumetofen controls wild-type as well as sdhBP225/H272R and sdhBP225H/H272 mutants. Pydiflumetofen does not control CG in the sdhBP225/H272Y mutant but is effective in the GTE control. Pydiflumetofen significantly controls Botrytis independently of the SdhB genotype in wounded berry assays. This condition resembles the berry cracking due to heavy rainfall right before harvest, as seen in recent years in the Chilean Central Valley. The findings demonstrate that pydiflumetofen effectively controls the grapevine Botrytis population, suggest a moderate risk of pydiflumetofen resistance, and highlight the significance of incorporating genetic data into the design of control programs.

PI4KIIIß Activity Regulates Lateral Root Formation Driven by Endocytic Trafficking to the Vacuole.

Lateral roots (LRs) increase the contact area of the root with the rhizosphere and thereby improve water and nutrient uptake from the soil. LRs are generated either via a developmentally controlled mechanism or through induction by external stimuli, such as water and nutrient availability. Auxin regulates LR organogenesis via transcriptional activation by an auxin complex receptor. Endocytic trafficking to the vacuole positively regulates LR organogenesis independently of the auxin complex receptor in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that phosphatidylinositol 4-phosphate (PI4P) biosynthesis regulated by the phosphatidylinositol 4-kinases PI4KIIIß1 and PI4KIIIß2 is essential for the LR organogenesis driven by endocytic trafficking to the vacuole. Stimulation with Sortin2, a biomodulator that promotes protein targeting to the vacuole, altered PI4P abundance at both the plasma membrane and endosomal compartments, a process dependent on PI4K activity. These findings suggest that endocytic trafficking to the vacuole regulated by the enzymatic activities of PI4KIIIß1 and PI4KIIIß2 participates in a mechanism independent of the auxin complex receptor that regulates LR organogenesis in Arabidopsis. Surprisingly, loss-of-function of PI4KIIIß1 and PI4KIIIß2 induced both LR primordium formation and endocytic trafficking toward the vacuole. This LR primordium induction was alleviated by exogenous PI4P, suggesting that PI4KIIIß1 and PI4KIIIß2 activity constitutively negatively regulates LR primordium formation. Overall, this research demonstrates a dual role of PI4KIIIß1 and PI4KIIIß2 in LR primordium formation in Arabidopsis.

First Report of canker and branch dieback of sweet cherry trees caused by Calosphaeria pulchella in Chile.

In Chile, the 2019-2020 sweet cherry season yielded 228,548 t, produced on 38,392 hectares and an average annual crop value about US$1.6 billion (http://www.iqonsulting.com/yb/). Between autumn 2019 and summer of 2020, branch and limbs dieback symptoms were observed in two 12-year-old sweet cherry (Prunus avium L.) orchards located in the O'Higgins region (Chile Central Valley). Furthermore, other symptoms such as wilting leaves, cankers, bark cracking, emission of gum exudates and internal wood necrosis were detected on trees of "Bing", "Santina" and "Sweetheart" cultivars (Cainelli et al. 2017). Wood fragments from symptomatic branches were surface sterilized with 95% ethanol, flaming and placed onto potato dextrose agar (PDA) amended with 0.5 g liter-1 of streptomycin sulfate (Berbegal et al. 2014). After 7 days of incubation at 25°C, pink to red colonies with white margins were isolated. Each isolate was characterized by having hyaline and oblong-ellipsoidal conidia of 5.76 ± 0.88 × 1.76 ± 0.36 µm (n=100) (Trouillas et al. 2012). According to these morphological features, the fungus was identified as Calosphaeria pulchella (Pers.: Fr.) J. Schröt (anamorph Calosphaeriosphora pulchella Réblová,L. Mostert, W. Gams & Crous) (Réblová et al. 2004). ITS (Internal Transcribed Spacer region of the rDNA) sequence comparison using BLAST analysis revealed a 99.48% identity and 100% query coverage between C. pulchella sequence HM237297 and the Chilean isolates. Moreover, the Chilean isolates were confirmed by means of phylogenetic analysis using ITS sequences of C. pulchella available in GenBank database. The maximum-parsimony phylogenetic tree supported the cluster analysis of the Chilean C. pulchella isolates with those obtained in other regions of the world with a bootstrap value of 95% (Berbegal et al. 2014; Trouillas et al. 2012). The Chilean ITS sequences were deposited into GenBank (MT378444 to MT378447). Two-year-old sweet cherry trees cv. Bing were inoculated with the Chilean isolates. Six trees were used as replicates. To accomplish this goal, two punctures of 5mm diameter were made in two branches per tree with a cork borer and a plug of mycelium from 7-day-old colonies was laid on the wound mycelium side down. Six trees were inoculated with sterile agar plugs. Every puncture was sealed with petroleum jelly and wrapped with parafilm. Four months after inoculation, the vascular streaking developing from the inoculated wounds was measured. The average lesion lengths on inoculated and non-inoculated shoots were 43.79 and 21.79 mm, respectively, which were significantly different according LSD Fisher test (p<0.05). C. pulchella was recovered from all the inoculated branches. No fungus was isolated from the controls, confirming Koch's postulates (Trouillas et al. 2012). To our knowledge this is the first report of C. pulchella causing canker and branch dieback in sweet cherry trees in Chile. This new disease represents a serious threat to the Chilean cherry industry, and further research on disease control is needed.

Botrytis prunorum Associated to Vitis vinifera Blossom Blight in Chile.

Table grapes are highly susceptible to Botrytis cinerea infections during the bloom period. After reaching the flower development stage, B. cinerea remains quiescent until berry ripening or gives rise to blossom blight under specific climate conditions. A research study was conducted on the Chilean Central Valley during the 2018-2019 growing season. Flowers of Vitis vinifera cv. Thompson Seedless were collected and B. cinerea was isolated together to a second and morphologically different species, characterized by white mycelium and low to no sporulation (11.4% of total isolates). Three randomly selected isolates within this population were genetically examined and identified as Botrytis prunorum based on a phylogenetic multilocus approach using partial regions of genes RPB2, HSP60, and G3PDH or NEP1 and NEP2. Pathogenicity tests showed that B. prunorum infects and causes wilting in healthy table grape flowers. B. prunorum isolates were able to infect Thompson Seedless berries, inducing lesions between 13.11 and 41.53% with respect to the lesion diameter generated by B. cinerea B05.10. The fungicide sensitivity was evaluated. The three genetically characterized isolates were sensitive to boscalid and to cyprodinil/fludioxonil mixture with a mean EC50 value of 5.5 µg/ml and 0.065 µg/ml, respectively. However, loss of sensitivity to fenhexamid was determined, with a mean EC50 value of 5.13 µg/ml. Our understanding about blossom blight in V. vinifera has been limited to B. cinerea. Here we associated B. prunorum as a second causal agent of this disease in Chile. This data represents a first approach to the epidemiological characteristics of B. prunorum associated with blossom blight in table grapes.

A Conservative Mutant Version of the Mrr1 Transcription Factor Correlates with Reduced Sensitivity to Fludioxonil in Botrytis cinerea.

Fludioxonil is a highly effective phenylpyrrole fungicide for controlling Botrytis cinerea. Although the field efficacy of fludioxonil remains high, Botrytis cinerea isolates with reduced sensitivity have been reported globally. The molecular target of fludioxonil still remains unknown; however, a mechanism of reduced sensitivity to fludioxonil underlies the overexpression of the ATP binding cassette (ABC) transporter AtrB in a dependent pathway of the Mrr1 transcription factor. Fludioxonil is a key player in controlling B. cinerea infection in table grapes in Chile. However, some isolates with a reduced sensitivity to fludioxonil were detected. This study observed endogenous atrB overexpression in Chilean isolates with reduced sensitivity to fludioxonil (n = 22) compared to the sensitive isolates (n = 10). All isolates increased the expression of atrB in a growth medium supplemented with fludioxonil (0.05 µg/mL). However, sensitive isolates showed lower atrB expression than those with reduced fludioxonil sensitivity. Remarkably, a mutant version of the transcription factor Mrr1 carrying 21 amino acid modifications was identified in all isolates with reduced sensitivity to fludioxonil. These changes alter the protein's transcription factor domain and the C-terminal portion of the protein but not the Zn (2)-C6 fungal-type DNA-binding domain. These results suggest a direct relationship between the conserved and divergent mutant version of mrr1 and sensitivity to fludioxonil. This study provides a new target for developing molecular diagnostic strategies to monitor B. cinerea's sensitivity to fludioxonil in the field.

Sucrose targets clathrin-mediated endocytosis kinetics supporting cell elongation in Arabidopsis thaliana.

Sucrose is a central regulator of plant growth and development, coordinating cell division and cell elongation according to the energy status of plants. Sucrose is known to stimulate bulk endocytosis in cultured cells; however, its physiological role has not been described to date. Our work shows that sucrose supplementation induces root cell elongation and endocytosis. Sucrose targets clathrin-mediated endocytosis (CME) in epidermal cells. Its presence decreases the abundance of both the clathrin coating complex and phosphatidylinositol 4,5-biphosphate at the plasma membrane, while increasing clathrin complex abundance in intracellular spaces. Sucrose decreases the plasma membrane residence time of the clathrin complex, indicating that it controls the kinetics of endocytic vesicle formation and internalization. CME regulation by sucrose is inducible and reversible; this on/off mechanism reveals an endocytosis-mediated mechanism for sensing plant energy status and signaling root elongation. The sucrose monosaccharide fructose also induces CME, while glucose and mannitol have no effect, demonstrating the specificity of the process. Overall, our data show that sucrose can mediate CME, which demonstrates that sucrose signaling for plant growth and development is dependent on endomembrane trafficking.

First Record of Colletotrichum anthrisci Causing Anthracnose on Avocado Fruits in Chile.

Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.

The Use of Drugs in the Study of Vacuole Morphology and Trafficking to the Vacuole in Arabidopsis thaliana.

Chemical compounds are useful to perturb biological functions in the same way as classical genetic approaches take advantage of mutations at the DNA level to perturb gene function. The use of bioactive chemicals currently called chemical genetic is especially valuable for cell biology. Chemical genetic approaches allow perturbations of cellular processes post-germination in a given time window controlling the severity of the effect by modifying or modulating the dose and/or the period of the treatment. Additionally, compounds can be applied directly to different mutants and translational fluorescent reporters/marker lines, expanding the repertoire of experimental setups addressing cell biology research. In this chapter, we describe standard protocols to visualize vacuole morphology and trafficking to the vacuole and the use of bioactive compounds as a proxy to study these biological processes.