Identification and distribution of species of Neofusicoccum that cause blueberry stem blight in Chile.

Stem blight is a destructive woody disease of blueberry (Vaccinium corymbosum) caused by several species of the family Botryosphaeriaceae. A field survey was conducted in the mayor blueberry production area of Chile, comprising latitudes 32°49'S to 40°55'S, to determine the occurrence and distribution of Botryosphaeriaceae in the region. Together, a multilocus analysis, morphological characterization, and phytopathogenicity testing were used to identify 51 Neofusicoccum isolates belonging to N. nonquaesitum (28 strains), N. parvum (22 strains), and N. australe (1 strain). Of these, N. parvum and N. nonquaesitum were the most commonly found, with N. parvum most frequent from latitude 37°40'S to the north and N. nonquaesitum predominantly located from the same latitude toward the south. Morphological traits of the isolates were consistent with the species identified by molecular techniques, despite the overlapping of conidial size of some isolates among species. Pathogenicity trials showed that the three species were pathogenic to blueberry plants and revealed that N. parvum and N. nonquaesitum were the most aggressive species, although variability in virulence was observed among isolates of N. parvum and N. nonquaesitum.

Regulatory Frameworks for the Access and Use of Genetic Resources in Latin America.

The Nagoya Protocol is a legal framework focused on the Access and Benefit Sharing of genetic resources, including Biological Control Agents. In order to comply with the Nagoya Protocol, countries in Latin America are establishing legal frameworks for access to genetic resources. Scientists face the challenges of the bureaucratic and administrative burden to obtain the access permits to study the biodiversity present in Latin American countries, which include the evaluation of biological control agents that can be used in sustainable production programs. In order to avoid the demotivation of scientists and students to work on biological control by blocking the opportunities to get new bioproducts, it is important to increase the communication between the regulatory authorities and the scientific community, to ensure the establishment of an effective structure and mechanisms to facilitate the process and reduce the time needed to obtain the access permits. On the other hand, the establishment of regional platforms for the exchange of information and harmonization of procedures can contribute to reinforce the collaboration among Latin American countries and facilitate regional studies and biocontrol activities. In this article, the legal framework in place in different countries in Latin America will be discussed and some possible solutions and ways forward to the major challenges observed will be presented.

First report of Colletotrichum fioriniae causing anthracnose fruit rot on Vaccinium corymbosum in Chile.

Vaccinium corymbosum L. is the most cultivated blueberry species in Chile. Chilean fruits typically take up to 50 days to reach oversea markets; therefore, controlling post-harvest pathogens is of outmost importance to maintain international food safety and quality standards. In February 2019, the Microbial Genetic Resources Bank at INIA received fruits of V. corymbosum cv. 'Brigitta Blue' from Mariquina (-39.567869, -72.992461), located in the southern Chilean blueberry production zone, for post-harvest disease diagnosis. Asymptomatic fruits were incubated in moist-chambers at 25 °C with light/darkness cycles of 12 h. After 5 d, some fruits showed sunken areas and small surface wounds that exudated orange masses of conidia; under the epidermis, gray acervuli were also detected. After 15d, these fruits became dehydrated, mummified, and covered by mycelia, all characteristic symptoms of anthracnose (Wharton and Schilder 2008). In Chile, Colletotrichum gloeosporioides has, thus far, been the only causal agent of anthracnose reported in blueberry (Lara et al. 2003). Conidia exudated from the diseased fruit were inoculated on potato-dextrose agar (PDA) and incubated at 25 °C for 7 d. The resulting colony was predominantly cottony with gray aerial mycelium, displaying masses of pale orange conidia; on the reverse side, the colony was a pink-reddish color. Under a microscope, conidia were hyaline, fusiform to elliptic in shape, and displaying guttulate of 12.2±1.2 × 4.17±0.3 µm (n=30), characteristics coinciding with those described for Colletotrichum fioriniae (Pennycook 2017; Shivas and Tan 2009) (Supplementary Figure 1). The isolate was deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) as RGM 3330. Genomic DNA extraction of RGM 3330 and phylogenetic analyses were carried out according to Cisterna-Oyarce et al. (2022). A multi-locus sequencing analysis was carried out using five genetic markers. The internal transcribed spacer (ITS), glyceraldehyde 3-phosphate dehydrogenase (gapdh), actin (act), and chitin synthase 1 (chs-1) were PCR-amplified following Damm et al. (2012) and -tubulin (tub) following Glass and Donaldson (1995). Sequences were deposited in GenBank (ON364141 for ITS and ON369167-70 for tub, act, chs-1, and gapdh, respectively) (Sayers et al. 2021). A BLAST analysis carried out in SequenceServer (Priyam et al. 2019), using a custom database of sequences retrieved from Damm et al. (2012) and Liu et al. (2020), showed that all genetic markers were 100% identical to those of C. fioriniae CBS 128517T (ITS (540/540 identities), gapdh (249/249), act (245/245), and chs-1 (274/274)), except for tub, which shared 99.8% of its identities (416/417) with this species. Maximum likelihood phylogenetic estimation clustered RGM 3330 with C. fioriniae strains CBS 128517T and CBS 126526 with 100% bootstrap support (Supplementary Figure 1). Koch's postulates were carried out with asymptomatic fruits of V. corymbosum cv. 'Brigitta Blue'. Prior to inoculation, fruits were surface-sterilized for 10 s in 70% ethanol, 3 s in 1% NaOCl, 10 s in 70% ethanol, rinsed three times with sterile distilled water, and subsequently placed in moist-chambers. Two groups of three repetitions of 20 fruits each were sprayed with 9 × 106 conidia/mL of RGM 3330 for the first group and with sterile distilled water for the control. After 5 d at 25 °C with light/darkness cycles of 12 h, only fruits sprayed with the conidial solution developed symptoms of anthracnose and the re-isolated fungi were identical in morphology to RGM 3330. This is the first report of C. fioriniae in blueberry in Chile. References Cisterna-Oyarce, V., Carrasco-Fernández, J., Castro, J. F., Santelices, C., Muñoz-Reyes, V., Millas, P., Buddie, A. G., and France, A. 2022. Gnomoniopsis smithogilvyi: identification, characterization and incidence of the main pathogen causing brown rot in postharvest sweet chestnut fruits (Castanea sativa) in Chile. Australasian Plant Disease Notes 17:2. Damm, U., Cannon, P. F., Woudenberg, J. H., and Crous, P. W. 2012. The Colletotrichum acutatum species complex. Stud. Mycol. 73:37-113. Glass, N. L., and Donaldson, G. C. 1995. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl. Environ. Microbiol. 61:1323-1330. Lara, O., Velazquez, C. G., and Ascencio, C. 2003. Colletotrichum gloeosporiodes in blueberry fruit. in: XIII Congreso de Fitopatología. Liu, X., Zheng, X., Khaskheli, M. I., Sun, X., Chang, X., and Gong, G. 2020. Identification of Colletotrichum species associated with blueberry anthracnose in Sichuan, China. Pathogens 9:718. Pennycook, S. 2017. Colletotrichum fioriniae comb. & stat. nov., resolving a nomenclatural muddle. Mycotaxon 132:149-152. Priyam, A., Woodcroft, B. J., Rai, V., Moghul, I., Munagala, A., Ter, F., Chowdhary, H., Pieniak, I., Maynard, L. J., Gibbins, M. A., Moon, H., Davis-Richardson, A., Uludag, M., Watson-Haigh, N. S., Challis, R., Nakamura, H., Favreau, E., Gómez, E. A., Pluskal, T., Leonard, G., Rumpf, W., and Wurm, Y. 2019. Sequenceserver: a modern graphical user interface for custom BLAST databases. Mol. Biol. Evol. 36:2922-2924. Sayers, E. W., Cavanaugh, M., Clark, K., Pruitt, K. D., Schoch, C. L., Sherry, S. T., and Karsch-Mizrachi, I. 2021. GenBank. Nucleic Acids Res. 49:D92-D96. Shivas, R. G., and Tan, Y. P. 2009. A taxonomic re-assessment of Colletotrichum acutatum, introducing C. fioriniae comb. et stat. nov. and C. simmondsii sp. nov. Fungal Divers. 39:111-122. Wharton, P., and Schilder, A. 2008. Novel infection strategies of Colletotrichum acutatum on ripe blueberry fruit. Plant Pathol. 57:122-134. Supplementary material Supplementary Figure 1: Isolation and identification of Colletotrichum fioriniae RGM 3330 from blueberry fruits cv. 'Brigitta Blue' from Chile. (A) A fruit showing anthracnose; (B) colony of Colletotrichum fioriniae RGM 3330 growing on PDA; (C) microscopic observation of the conidia (100x magnification; bar=10 µm); (D) phylogenetic tree resulting from a maximum likelihood analysis of combined sequence data from ITS, act, chs-1, gapdh, and tub regions for Colletotrichum acutatum species complex, number in the nodes represent ultrafast bootstrap values.

Silverleaf (Chondrostereum purpureum) Effects on Japanese Plum (Prunus salicina).

Silverleaf is an important fungal trunk disease of fruit crops, such as Japanese plum (Prunus salicina). It is known that infection by Chondrostereum purpureum results in discolored wood, "silvered" foliage, and tree decline. However, effects on fruit yield and quality have not been assessed. Therefore, the objectives of this study were to determine C. purpureum pathogenicity on P. salicina and the effects on physiology, fruit yield, and quality, in Chile, in 2019 and 2020. Wood samples from affected plum trees were collected in the Chilean plum productive area. Fungi were isolated by plating wood sections from the necrosis margin on culture media. Morphological and molecular characteristics of the isolates corresponded to C. purpureum (98%). Representative isolates were inoculated from healthy plum plants and after 65-d incubation, wood necrotic lesions and silver leaves were visible. Fungi were reisolated, fulfilling Koch's postulates. To determine Silverleaf effects, xylem water potential and fruit yield and quality were measured in healthy and Silverleaf-diseased plum trees 'Angeleno'. Water potential was altered in diseased trees, and fruit yield was reduced by 51% (2019) and by 41% (2020) compared to fruit from healthy trees. Moreover, cover-colour, equatorial-diameter, and weight were reduced, and fruit were softer, failing to meet the criteria to be properly commercialized and exported to demanding markets.

Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum.

BACKGROUND: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops. RESULT: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation. CONCLUSIONS: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

First report of Eutypa lata causing cankers and dieback in sweet cherry in Chile.

Sweet cherry (Prunus avium) is one the most important fruit crops in Chile. Its production has significantly grown in recent years, reaching 228,448 tons exported in 2019/2020, to 47 countries. One of the main threats for this expanding crop are fungal pathogens, especially those that cause wood diseases. Cherry orchards (n=35) located in the central area of Chile, from Curicó (34°58'58''S 71°14.366'W) to Angol (37°47'42.7''S 72°42.982'W), were surveyed during 2020. Wood samples were collected (n= 72) from living branches and trunks showing dieback, cankers and dark necrosis, mostly wedge shaped. Small wood sections (0.5-cm) were cut off from the margin of the necrosis and surface disinfected using 0.5% v/v sodium hypochlorite. Sections were plated on a quarter-strength potato dextrose agar amended with 1mg/L tetracycline (PDA-tet). Plates were incubated at 25°C until mycelial development and subsequently the isolates were purified transferring excised fungal tips to PDA. Colonies (n=21) developed white cottony mycelia, which turned slightly greyish and flatter after 10-days at 25°C. Isolates developed black pycnidia which released beige conidial matrixes after subsequent 15-days at 25 +/-2°C and 12-h photoperiod. Conidia were hyaline, curved and filiform, measuring 19.8-(27.9)-36.7 µm length (lineal) x 1.2-(1.7)-1.9 µm width (n=70), according to Eutypa lata (Rappaz, 1984). DNA was extracted from mycelia of the representative isolates HMCe30a, HMCe41a, HMCe109c and HMCe110a. The partial ß-tubulin gene was amplified using bt2A/bt2B primers (Glass & Donaldson 1995) and the internal transcribed spacer region was amplified using ITS1/ITS4 primers (White et al. 1990). Sequences were BLAST analyzed, finding that ITS shared 99% and ßTUB 100% identity with E. lata strain CBS 208.87 (Rolshausen et al. 2006). Sequences were accessioned to GenBank (MW363035, MW363034, MW363033 and MW363032 [ITS], and MW366820, MW366819, MW366818and MW366817 [ßTUB]). The isolates were inoculated on sweet cherry healthy plants cv. Kordia, produced by rooting scions in tap water amended with 500 ppm of indole-butyric acid, for 30 days. An injury was made in the upper third of the shoot using a sterile 0.5-cm diameter corkborer. Mycelial plugs were placed on the injuries and covered with plastic film, using sterile agar for controls (n=25). Plants were incubated in aerated tap water for 60 days at 23 +/-3 °C. After incubation, plants were cut exposing dark-brown necrotic lesions, while control plants remained asymptomatic. Moreover, 2-year old potted plants cv. Lapins were inoculated (n=3 per isolate) with mycelial plugs, on fresh cuts of their main lateral branches, in January 20th, and remained under partial shade for 72-days. After incubation, bark was removed from inoculated branches and the necrotic lesions length was measured. HMCe109c was the most virulent isolate (3.6 cm), followed by HMCe30a (2.1 cm), HMCe41a (1.9 cm) and HMCe110a (1.1 cm), while symptoms were not reproduced in controls. Fulfilling Koch's postulates, fungi were reisolated from all inoculated plants in both pathogenicity tests and no fungus was recovered from controls. To our knowledge this is the first report of Eutypa lata causing wood decay in sweet cherry in Chile. The pathogen was recently reported causing dieback of grapevines in Chile (Lolas et al. 2020). These are significant findings due to the frequent proximity of sweet cherry orchards and vineyards, which facilitates cross infections.

First report of Seimatosporium vitifusiforme causing trunk disease in Chilean grapevines (Vitis vinifera).

Grapevine is one of the most important fruit crops in Chile and trunk diseases reduce the productivity, quality, and longevity of the vineyards. A survey was conducted in ancient (> 50 years) vineyards of Cauquenes (35°57´14´´S 72°17´07´´W) and Itata valleys (36°38´13´´S 72°30´57´´W), located in the central area of Chile, during 2019. Trunks and cordons showing dieback and dark brown to black wood discoloration were collected from 50 to 200-year-old plants of six cultivars: País, Moscatel, Torontel Amarilla, Carignan, Aliatica and Aligote. The bark was removed and 0.5-cm sections were cut from the edges of necrotic wood lesions. Subsequently, pieces were surface disinfected using 10% v/v sodium hypochlorite bleach (4.9% chlorine), plated on acidified quarter-strength potato dextrose agar (APDA) (25% PDA, acidified with 0.1% v/v 85% lactic acid) and incubated at 25°C, for 14 to 28 days. Hyphal tips were excised and transferred to PDA to obtain pure cultures. Along with the conidiomata and conidia produced, growth rate, color and shape of the colonies on PDA, after 7 and 14 days of incubation at 25°C (n=17), were recorded. DNA was extracted from pure cultures of three isolates on PDA: HMV3, HMV64 and HMV81. The internal transcribed spacer region and partial ß-tubulin genes were amplified, using ITS1/ITS4 (White et al. 1990) and bt2A/bt2B (Glass & Donaldson 1995) primers, respectively. Sequences were subjected to NCBI BLAST search and compared to the published sequences. Isolated colonies were whitish to light-brown, cottony with a smooth margin (n=37). Their mycelium grew 1.9 cm after 7-days and 3.2 cm after 14-days of incubation on PDA, at 25°C. Colonies produced black globose pycnidia and curved, slightly-pigmentated, three-septated conidia 22.3-(29.8)-32.2 x 3.9-(4.8)-5.3 µm (n=30), with apical and basal flexuous appendages 4.3-(12.7)-21.5 µm (n=20). When compared to type sequences of Seimatosporium vitifusiforme (Lawrence et al. 2018), ITS and ßtub sequences identity of these isolates were 99 to 100% identical. To produce uniform healthy plants for pathogenicity tests, Petit Syrah canes (1-year old) were rooted in tap water amended with 500 ppm of indole-butyric acid, for 30 days. Plants were inoculated with 0.5-cm diameter mycelial plugs of actively growing colonies of the isolates HMV3, HMV64 and HMV81 (GenBank accessions no. MW026664, MW048518; MW026665, MW048519, and MW026666, MW048520, respectively). Sterile agar plugs were used for controls. Five plants per pathogen isolate were incubated at 25°C, in a humid chamber, for 25 days, and seven additional plants per isolate were incubated in aerated tap water, for 55 days. After the incubation period, the bark was removed and the lesions were measured. Dark necrotic lesions identical to the original observations were reproduced, both in the high humidity chamber (6% length) and water (10% length). There were no differences in lesion length among the isolates (P < 0.05). Control vines remained asymptomatic. To fulfill Koch´s postulates, isolations were made from symptomatic vines and compared to the ones used for inoculation, and found to be identical. Seimatosporium vitifusiforme was previously reported as a pathogen of Vitis vinifera in California, USA (Lawrence et al. 2018). Consequently, this is the second report of this fungus as a grapevine pathogen and the first one affecting Latin-American grapevines.