Diversity and functional assessment of indigenous culturable bacteria inhabiting fine-flavor cacao rhizosphere: Uncovering antagonistic potential against Moniliophthora roreri.

The Peruvian Amazonian native cacao faces ongoing challenges that significantly undermine its productivity. Among them, frosty pod rot disease and cadmium accumulation result in losses that need for effective and environmentally safe strategies, such as those based on bacteria. To explore the biological resources in the cacao soil, a descriptive study was conducted to assess the diversity of culturable bacteria across three production districts in the Amazonas region: La Peca, Imaza, and Cajaruro. The study also focused on the functional properties of these bacteria, particularly those related to the major issues limiting cacao cultivation. For this purpose, 90 native bacterial isolates were obtained from the cacao rhizosphere. According to diversity analysis, the community was composed of 19 bacterial genera, with a dominance of the Bacillaceae family and variable distribution among the districts. This variability was statistically supported by the PCoA plots and is related to the pH of the soil environment. The functional assessment revealed that 56.8% of the isolates showed an antagonism index greater than 75% after 7 days of confrontation. After 15 days of confrontation with Moniliophthora roreri, 68.2% of the bacterial population demonstrated this attribute. This capability was primarily exhibited by Bacillus strains. On the other hand, only 4.5% were capable of removing cadmium, highlighting the biocontrol potential of the bacterial community. In addition, some isolates produced siderophores (13.63%), solubilized phosphate (20.45%), and solubilized zinc (4.5%). Interestingly, these traits showed an uneven distribution, which correlated with the divergence found by the beta diversity. Our results revealed a diverse bacterial community inhabiting the Amazonian cacao rhizosphere, showcasing crucial functional properties related to the biocontrol of M. roreri. The information generated serves as a significant resource for the development of further biotechnological tools that can be applied to native Amazonian cacao.

Uromyces savulescui on statice (Limonium sinuatum) in the Americas and reevaluation of its reported geographic range.

Limonium sinuatum (Plumbaginaceae) is the most commonly cultivated recognizable cut flower crop in the genus Limonium. It is known by several common names including statice and sea lavender, due to its lilac-colored flowers and the fact that it naturally inhabits mainly coastal areas (Mellesse et al, 2013). Limonium sinuatum is native to the Mediterranean, although as a popular garden plant, has been naturalized in other parts of the world including coastal areas of California (USDA NRCS 2020). Cultivated L. sinuatum is used in fresh and dry flower arrangements in the Americas, comprising approximately 20% of the floriculture cultivated area in Ecuador (Vega and Morales 2011; Abascal Cañas 2017). In December 2014, L. sinuatum plants in the public park "Baños del Inca" in Cajamarca, Peru (S 7 9'46"; W 78 27'53"), were found infected with a rust disease. The plants were scattered in the park but infection incidence was 100% as individual plants were all found to be infected (Fig 1). Based on the percentage of symptomatic areas, including the yellow halos around pustules, calculated with ImageJ (Collins, 2007) from field photographs, the disease severity was estimated to be 58.9% in average, ranging from 19.8% up to 90.0%. Uredinia were present on both sides of the leaves as well as on stems and were roundish, oblong, pulverulent, and cinnamon brown in color; urediniospores 25.5 to 35.0 × 22.5 to 31.0 µm, were globoid to ellipsoid; urediniospore walls were cinnamon-brown, 2.5 to 3.0 µm thick, densely verrucose, with 2 to 3 equatorial germ pores. Few telia were present on leaves; these were scattered roundish or oblong, and greyish in color; teliospores 26.5 to 41.0 × 16.0 to 25.0 µm, were ellipsoid to obovoid, mostly attenuated at the apex; teliospore walls were colorless, 2-3 µm thick at sides, and up to 10 µm thick at apex. Teliospores readily germinated in sori producing basidia and basidiospores (Fig. 2). The rust features and dimensions of rust spores are consistent with available descriptions of Uromyces savulescui Rayss (Guyot 1951; Vakalounakis and Malathrakis 1987). To confirm identity, a 576 bp region of the 28S subunit of the ribosomal DNA repeat was sequenced following previously published protocols and primers (Aime 2006, Aime et al. 2018). The resulting sequence (GenBank Accession No. OR291160) shared 99.83% (573/574 bp) identity with a sequence deposited as Uromyces limonii (DC.) Lev. (accession KY764194, BPI910295, Demers et al. unpublished) from L. sinuatum in Ethiopia. However, U. limonii produces orange uredinia, thin-walled yellow-orange urediniospores, teliospores with mostly light chestnut brown wall and infects different hosts (Savile and Conners 1951). It is likely that KY764194 represents a misannotated record of U. savulescui. While Koch's postulates can be a useful tool for establishing causality in certain infectious diseases, their use may be limited when it comes to rust diseases based on old herbarium specimens. In our case, due to the age of the specimen, which is almost nine years old, various other methods were employed to identify the pathogen. These methods included microscopic examination for morphological criteria of the urediniospores and teliospores, as well as molecular techniques like 28S rDNA sequencing. Rust disease on L. sinuatum has been previously reported in Ecuador but the causal agent was identified as a Puccinia sp. and reported that the rust was able to destroy entire plots in humid conditions (Vega and Morales 2011). Whether this report also represent U. savulescui is not certain, but given that the urediniospores of Puccinia species are generally 2-celled, it is unlikely. García-Hernández et al. (2008) reported U. limonii on Limonium spp. from Chile, and Coca (2020) also reported U. limonii on Limonium sp., from Bolivia. However, judging from the photomicrographs (Coca 2020), the rust in the latter report is definitely U. savulescui and not U. limonii. Uromyces savulescui has been previously reported from the Mediterranean region and the Canary Islands (Vakalounakis and Malathrakis 1987). To our knowledge there is no report of this rust in the Americas, excepting the probable misidentifications already listed herein. The specimen has been deposited in the Arthur Fungarium at Purdue University as PURN15037.

Morphological, phylogenetic, and genomic evidence reveals the causal agent of thread blight disease of cacao in Peru is a new species of Marasmius in the section Neosessiles, Marasmius infestans sp. nov.

The thread blight disease (TBD) of cacao ( Theobroma cacao) in the department of Amazonas, Peru was recently reported to be caused by Marasmius tenuissimus (sect. Neosessiles). This same species is known to be the main causal agent of TBD in West Africa. However, some morphological characteristics, such as the presence of rhizomorphs, the almost exclusively white color, and pileus sizes less than 5 mm, among others, differ to the description of M. tenuissimus. Therefore, we aimed to conduct a taxonomic revision of the cacao-TBD causal agent in Peru, by using thorough micro and macro morphological, phylogenetic, and nuclear and mitochondrial genomic approaches. We showed that the causal agent of TBD of cacao in Amazonas, Peru, belongs to a new species, Marasmius infestans sp. nov. This study enriches our knowledge of species in the sect. Neosessiles, and strongly suggests that the M. tenuissimus species complex is highly diverse.

First Report of Anthracnose Caused by Colletotrichum asianum on Mango (Mangifera indica) in Peru.

Mangoes (Mangifera indica L.) are one of the most important export fruits in Peru and anthracnose, caused by several species in the Colletotrichum gloeosporioides species complex (CGSC), is one of their main postharvest diseases (Alvarez et al. 2020). Balsas is the major mango producing district in the Amazonas department, where farmers practice intercropping in orchards mostly of less than 5 ha (Cabezudo Cerpa 2022). In July 2021, mango fruits cv. Kent with anthracnose were detected at an incidence of 55 to 80% during postharvest in Balsas. Symptoms included sunken dark brown lesions with appearance of orange conidiomata at advanced stages of the disease. We collected two samples of infected mangoes from a farm located at 6°51'01" S, 77°59'48" W (1088 m.a.s.l.). One axenic culture (INDES-AM1) was obtained from a hyphal tip of a monosporic colony and cultivated on PDA medium at 25 °C in the dark. The growing rate of the colony was 8.1 mm.day-1. Conidia were hyaline, guttulate, unicellular and cylindrical with narrowing center, with dimensions of 15.8 to 23.5 × 4.5 to 7.6 µm (mean = 18.6 ± 0.03 × 6.0 ± 0.02 µm, SE, n = 50), consistent to the CGSC (Weir et al. 2012). Appressoria were dark brown, and ovoid to slightly irregular in shape, ranging from 5.3 to 10.1 × 4.7 to 8.3 µm (mean = 7.9 ± 0.02 × 6.0 ± 0.02 µm, SE, n = 50). Koch's postulates were fulfilled on mature mango fruits of the same cultivar and from the same district. Mangoes were washed with detergent and left to dry before inoculation. PDA-mycelial plugs of 0.5 cm wide were transferred on two different locations of two fruits, with four replicates. One location was previously wounded with five needle punctures of 3 mm depth. The inoculated fruits were maintained in a moist chamber at ambient light and temperature (18.9 ± 0.5 °C, SE). Symptoms appeared three-to-five days post inoculation (dpi), and the superficial diameter of the lesions were 8.3 ± 1.5 and 3.6 ± 2 mm with the invasive and the superficial inoculation approaches, respectively, at five dpi. Lesions were very similar to original symptoms. Macro and micromorphological characteristics of the re-isolated fungal colonies were the same as isolate INDES-AM1. Molecular identification of the pathogen was carried out following Weir et al. (2012). Total DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega Corp., Madison, Wisconsin) and the ribosomal internal transcribed spacer (ITS), and partial sequences of the chitin synthase (CHS1), actin (ACT), ß-tubulin 2 (TUB2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear genes were sequenced (Accession numbers: OP425395, OP440444, OP440442, OP440443, OP555062, OP555063). ITS, CHS1, ACT, TUB2, CAL, and GAPDH sequences were 98.6, 100, 100, 99.5, 100, and 99.08% identical to Colletotrichum asianum type strain ICMP 18580 sequences, respectively. Additionally, a bootstrapped maximum likelihood midpoint-rooted phylogeny with a multilocus dataset with the six sequences from reference strains of C. asianum and closely related species within the CGSC revealed that strain INDES-AM1 is C. asianum. This species has been found causing anthracnose on M. indica in at least 15 different countries in Africa, America, Asia, and Oceania (Weir et al. 2012). It was originally described from coffee and has multiple other hosts (Prihastuti et al. 2009; Lima et al. 2015). To the best of our knowledge, this is the first report of C. asianum infecting mangoes in Peru.

First Report of Thread Blight Caused by Marasmius tenuissimus on Cacao (Theobroma cacao) in Peru.

Peru is the second largest producer of organic cocoa and one of the most important suppliers of fine aroma cocoa beans in the world (Sánchez et al. 2019). The fine aroma cocoa produced by smallholder farmers in the Bagua and Utcubamba Provinces, Amazonas Department, under the name of "Cacao Amazonas Peru", is protected by the Peruvian appellation rules (Díaz-Valderrama et al. 2020). Despite this importance, native diseases of the crop (Theobroma cacao) are poorly documented due to difficulty of access in this region. In November 2020 we conducted expeditions into Imaza District (4°47'09.4"S 78°16'51.6"W), a significant producer of fine aroma cocoa in terms of number of cultivated plots (4,651 out of 6,505 total in the Bagua Province) (INEI 2012). We visited 20 farms of < 2-ha in size; in 19 of these small farms, T. cacao trees were found infected with a white fungal thread blight and rhizomorphs covering branches and leaves. Disease incidence ranged from 90 to nearly 100%, and severity exceeded 80% on the eight farms with the most deficient phytosanitary management. Heavily infected leaves were hanging on branches by mycelial threads, harboring tiny (0.5 to 5.3 mm broad) white mushrooms. These symptoms and signs correspond to the thread blight disease constellation (TBD) of cacao caused by various species of Marasmius and Marasmiellus (Amoako-Attah et al. 2020). Mushrooms lacked a collarium, and their stipes were absent or rudimentary (< 2-mm long) and eccentric, consistent with Marasmius tenuissimus (Tan et al. 2009). Axenic cultures were obtained by surface sterilization of mycelium threads with 2% NaClO, rinsed three times in sterile water, plated on potato dextrose agar medium (PDA), and incubated for 7 days at 25°C. Hyphae was non-pigmented with clamp connections, consistent with the genus Marasmius. We extracted the DNA of isolate INDES-AFHP31 using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin) and sequenced the rDNA internal transcribed spacer 1 and 2 intervening the 5.8S subunit (ITS), and the 28S subunit (LSU) (Accession numbers: OM720123 and OM720135) according to Aime and Phillips-Mora (2005). The ITS and LSU sequences were 97.92 to 98.79% and 99.07 to 99.30% identical, respectively, with published sequences from M. tenuissimus from Ghana (Amoako-Attah et al. 2020). The pathogenicity test was conducted by inoculation of ten healthy cacao leaves with 7-day-old mycelium PDA discs of isolate INDES-AFHP31. An equal number of healthy cacao leaves were inoculated with PDA discs without mycelium as control. The observation of TBD symptoms and signs in the non-control set of cacao leaves starting at 3 days post inoculation, and the re-isolation of the same fungus from infected tissue confirmed its pathogenicity on cacao. Isolate INDES-AFHP31 was deposited as a dried culture into the herbarium Kuélap of the Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas (voucher KUELAP-2251). Marasmius tenuissimus was originally reported from dead and living twigs and leaves of unidentified dicotyledonous trees from Indonesia, Brazil, and Bolivia (Singer 1976). However, it was first associated with TBD of cacao in Ghana in 2020, being the most frequently TBD-causing fungus isolated in the country (Amoako-Attah et al. 2020). Its discovery in 19 of the 20 surveyed cacao farms in Imaza District, Amazonas, Peru, reveals its importance as a cacao pathogen in the Western hemisphere.

First report of Paramyrothecium roridum causing leaf spot and stem rot diseases on Coffea arabica in Peru.

Peru is the ninth exporter of coffee (Coffea arabica) in the world, and Amazonas is among its most important producing departments (INIA 2019). In July 2021, in the nursery of the "Instituto de Investigación para el Desarrollo Sustentable de Ceja de Selva", in Huambo district (6° 26' 11.19'' S; 77° 31' 24.18'' W), four-month-old coffee seedlings cv. Catimor with 0.5-2.0 cm brown concentric leaf spots and rotten stems, bearing white mycelial tufts and black sporodochia, were observed at 30% incidence. Infected seedlings were collected. Foliar sections of 2-3 mm with infected tissue were surfaced disinfected in 2% NaClO and transferred onto Petri plates containing potato dextrose agar medium (PDA). The plates were incubated at 25° C for 7 days. We obtained three isolates (INDES-AFHP61, INDES-AFHP62 and INDES-AFHP66) with similar morphology from different seedlings. Colonies (16-17 mm diam.) formed concentric rings with white aerial mycelium, giving rise to viscous and olivaceous dark green sporodochial conidiomata. Conidia (4.82-5.77 × 1.34-1.65 µm; n = 30) were cylindric, hyaline, smooth, and aseptate. These morphological features correspond to Paramyrothecium spp. (Lombard et al. 2016). The DNA of isolates was extracted using the Wizard® Purification Kit (Promega Corp., Madison, Wisconsin), and the internal transcribed spacer 1 and 2 intervening the 5.8S subunit rDNA region (Accession numbers: OM892830 to OM892832), and part of the second-largest subunit of the RNA polymerase II, the calmodulin and the ß-tubulin genes (OM919453 to OM919461) were sequenced following Lombard et al. (2016). All sequences had a percent identity greater than or equal to 99% to corresponding sequences of the P. roridum type specimen (CBS 357.89). Additionally, a multilocus Maximum Likelihood phylogenetic analysis incorporating sequence data from previous relevant studies (Lombard et al. 2016; Pinruan et al. 2022) grouped our three isolates together with the type and other specimens of P. roridum in a strongly supported clade, confirming the species identification. To evaluate pathogenicity, four-month-old coffee seedlings cv. Catimor were sprayed with 10 mL of conidial suspensions at 1 x 106 /mL. A set of control seedlings were inoculated with sterile water. Seedlings were maintained in a humidity chamber at 25 °C. After 15 days brown concentric foliar spots, stem rotting, mycelial tufts and sporodochia (same symptoms and signs observed originally at the nursery) arose in the non-control seedlings. The pathogen was re-isolated on PDA, confirming P. roridum was the causal agent of leaf spot and stem rot diseases of coffee. Paramyrothecium roridum has wide geographic distribution and host range (Lombard et al. 2016). This pathogen was reported to infect C. arabica in Mexico and Coffea sp. in Colombia (Pelayo-Sánchez et al. 2017; Lombard et al. 2016; Huaman et al. 2021). It was also reported in Africa infecting soybeans (Haudenshield et al. 2018), in Brazil infecting Tectona grandis (Borges et al. 2018), in Egypt infecting strawberries (Soliman 2020), and in Malaysia infecting Eichhornia crassipes (Hassan et al. 2021). To the best of our knowledge, this is the first time P. roridum is reported on coffee in Peru.

First report of Stemphylium leaf spot caused by Stemphylium vesicarium on alfalfa (Medicago sativa) in Peru.

Alfalfa (Medicago sativa) is the most cultivated fodder crop in Peru with 172,000 ha cultivated (MINAM 2019), and Arequipa is the top producing region with 40% of the national production in 2015 (Santamaría et al. 2016). In January-April 2019 (av. 20°C and 70% RH), most alfalfa fields in Majes-Pedregal, Arequipa were affected by an unidentified foliar disease. One of the fields was located at the farm of the Universidad Nacional de San Agustín de Arequipa (16°19'29.6" S, 72°12'59.9" W). Symptoms appeared as elliptical light brown spots witdark brown borders (Fig. S1a and b). The field (~60 × 60 m) was divided into ~30 × 12 m sections and two plants in each section were collected (20 plants total). Plants were digitized and the leaflet diseased area was calculated with ImageJ 1.53a, from which an incidence of 100% and a severity of 38.7 ± 4.4 % were estimated. Microscopical observations at the leaflet spots revealed consistently the presence of oblong multiseptated conidia (23.6-42.8 × 16.5-25.2 µm; av. 33.3 × 20.9 µm; n = 40) of the genus Stemphylium (Simmons 1969; Woudenberg et al. 2017) (Fig. S1c). We obtained 10 pure cultures by placing conidia from the spots directly onto potato dextrose agar medium with the aid of stereoscope and sterile forceps. Two isolates (UNSA-StemV01 and UNSA-StemV02) were incubated further until ascospore production at room temperature with no special light stimulus. After 45 days of growth, globose pseudothecia and ellipsoidal ascospores (25.4-38.7 × 11.2-16.6 µm; av. 31.9 × 13.7 µm; n = 30) formation occurred (Fig. S1d and e). We extracted the DNA from these two isolates using Wizard® Purification Kit (Promega Corp., Madison, WI) and sequenced the internal transcribed spacer 1 and 2 intervening 5.8S rDNA subunit (GenBank accessions: MT371236-37), and the glyceraldehyde-3-phosphate dehydrogenase (MT375513-14) and the calmodulin (MT375515-16) genes, highly resolutive markers to identify Stemphylium species, following Woudenberg et al. (2017). We retrieved sequence data available from 43 isolates of nine Stemphylium species (Han et al. 2019; Woudenberg et al. 2017), and built a mid-point rooted phylogeny with the three-loci concatenated data set (Fig. S2). We identified our isolates as S. vesicarium (Fig. S2). Koch's postulates were fulfilled by spray-inoculation with conidia from isolate UNSA-StemV01 suspended in sterile water (1×104 / mL) to two healthy 50-day old alfalfa plants growing on pots in the university greenhouse (av. 25°C and 70% RH). Two plants sprayed with sterile water without conidia served as control. Symptoms appeared after 21 days of inoculation, and when conidia were re-isolated, they were the same as originally obtained. No symptoms developed in the control plants. This confirmed that S. vesicarium is the causal agent of the alfalfa disease in Majes-Pedregal, identified as Stemphylium leaf spot. revious studies documented S. vesicarium on asparagus and onion in Peru (Castillo Valiente 2018; Vásquez Salas 2018; Vásquez Sangay 2013), but molecular characterization has only been applied to S. lycopersici from potatoes (Woudenberg et al. 2017). Stemphylium vesicarium has been documented in various crops, including alfalfa, and countries in Europe, North America, Africa, Asia and in Australia and New Zealand (Han et al. 2019; Woudenberg et al. 2017). This occurrence is the first report of S. vesicarium on alfalfa in Peru. The disease compromises the quality of this fodder crop, so actions need to be taken in Arequipa.