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Occurrence of Anthracnose Fruit-rot on the Endangered Plant Species Harrisia portoricensis
Casiani Soto-Ramos and Merari Feliciano-Rivera

Caribbean Naturalist, No. 56 (2019)

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Caribbean Naturalist 1 C. Soto-Ramos and M. Feliciano-Rivera 22001199 CARIBBEAN NATURALIST No. 56N:1o–. 1516 Occurrence of Anthracnose Fruit-rot on the Endangered Plant Species Harrisia portoricensis Casiani Soto-Ramos1 and Merari Feliciano-Rivera1,* Abstract - Harrisia portoricencis (Higo Chumbo) is a columnar cactus that belongs to the Cactaceae family. This species is endemic to Puerto Rico and is restricted to 3 islands: Mona, Monito, and Desecheo. Since 2014, individuals from the H. portoricensis collection at the University of Puerto Rico-Mayagüez Campus showed fruit-rot symptoms. Thus, the objective of our research was to characterize the causal agent of the fruit-rot symptoms on H. portoricensis. We based our identification of 4 fungal isolates (HARP 15-01, HARP 15-02, HARP 15-03, and HARP 15-04) on morphology, pathogenicity, and molecular characteristics of the internal transcribed spacer region (ITS) and partial sequences of the β-tubulin (TUB2) genes. The phenotypic appearance of all isolates on potato dextrose agar (PDA), size of the conidia and appressoria matched with the previous description of Colletotrichum siamense. All isolates were pathogenic to H. portoricensis with different levels of severity, ranging from moderate to severe fruit-rot damage of colonized tissue after 10 d of incubation. The partial sequences of the β-tubulin gene of the 4 isolates showed 99% homology with reference sequences C. siamense from the GeneBank. To our knowledge, this is the first report of C. siamense causing fruit rot in the endangered plant species H. portoricensis. Introduction The Cactaceae is a diverse family with 53 genera and 407 accepted taxa that commonly survive in water-scarce environments (Godinez-Álvarez et al. 2003, Nobel 2002, Rojas-Sandoval and Meléndez-Ackerman 2011). Despite its broad diversity, many species are classified as vulnerable, threatened, and endangered (Ortega-Baes and Godinez-Álvarez 2006, Walter and Gillett 1998). The genus Harrisia, which is native to South America and the Caribbean with 18 accepted species, is within this conspicuous and ecologically important family (Fleming and Valiente-Banuet 2002, Nobel 2002, Rojas-Sandoval and Meléndez-Ackerman 2011). Harrisia portoricensis Britt. (Higo Chumbo) is in the order Caryophyllales and endemic to Puerto Rico (USDA 2018). Harrisia portoricensis is branchless, has a columnar shape, and can reach up to 1.83 m (6 ft) in height. It has greenishwhite hermaphroditic flowers that produce a strong smell and large amounts of pollen and nectar (Liogier 1994, Proctor 1984, Rojas-Sandoval and Meléndez- Ackerman 2009). The fruits are round, yellow-colored, and thornless, and possess over 1500 black seeds protected by a translucent pulp (Rojas-Sandoval and Meléndez-Ackerman 2011). After anthesis, the fruits develop over 25–30 d, after which fruits remain as potential seed dispersers for 2 months (Rojas-Sandoval and Meléndez-Ackerman 2011). Harrisia portoricensis is protected under the 1Department of Agro-environmental Sciences, University of Puerto Rico, Mayagüez, PR 00680, USA. *Corresponding author - merari.feliciano@upr.edu. Manuscript Editor: Noris Salazar Caribbean Naturalist C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 2 Endangered Species Act of 1973. Since 1990, this species has been considered an “extinct species” (USFWS 1990) and has not been found in areas of Puerto Rico except the small semi-arid islands Mona, Monito, and Desecheo (Rojas-Sandoval, 2010, USFWS 1990). The most abundant population of H. portoricensis is on Mona Island with 59,857 individuals (Rojas-Sandoval 2010, Rojas-Sandoval and Meléndez-Ackerman, 2013a). Biotic and abiotic factors are potential threats to their conservation. Among them, Sus scrofa L. (Wild Boar), Capra hircus L. (Feral Goat), and Panicum maximum Jacq. [= Megatyrsus maximum] (Guinea Grass) are potential threats to the conservation of H. portoricensis on Mona Island (Rojas-Sandoval and Meléndez-Ackerman 2012, 2013b). Furthermore, the species’ narrow genetic variability results in genetic vulnerability to resist attacks by invasive species, pests, and pathogens. Stem symptoms such as circular necrotic lesions, circular brown spots, and tunnels have been observed in H. portoricensis in their natural habitat at Mona Island, but the causal agent has not been described (Rojas-Sandoval and Meléndez 2013b). Fruit symptoms have not been reported on Mona Island. However, in a nursery collection of 22 individuals at the University of Puerto Rico, Mayagüez Campus, fruit-rot symptoms have been commonly observed since 2014. One of the major goals of the conservation and recovery plans of endangered plant species is to identify the factors limiting the distribution and abundance of the species including biotic and abiotic factors. Thus, the objectives of our research were to (1) isolate the causal agent of fruit-rot symptoms, (2) describe its pathogenicity, and (3) complete morphological and molecular descriptions of the causal agent. Methods Collection of samples We evaluated 22 individuals of H. portoricensis for fruit-rot symptoms. We collected symptomatic fruits and stored them in plastic bags on ice at the Plant Pathology and Epidemiology Lab of the Agricultural Experiment Station of Isabela, PR, USA, for further detailed description of signs and symptoms . Fungal isolates Symptomatic tissue resembled fungal damage; thus, the methods we used for the isolation were specific for fungi. We collected three 5 × 5 mm2 pieces of tissue from the margins of infected fruit tissue, which we surface-sterilized by placing in 1% sodium hypochlorite for 1 min and then rinsing 3 times with sterile water. We placed the sterilized samples on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) acidified with 85% lactic acid and incubated the tissue at 27 °C for 7 d (Agrios 2005). After the incubation period, we re-cultivated the fungi on green bean agar (397 g fresh green beans plus 20 g of agar for 1 L artificial media) for purification and conidia production. After the fungi were purified, we obtained single-conidia cultures for the pathogenic test and morphological and molecular characterization using the procedure described by Choi et al. (1999). Caribbean Naturalist 3 C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 Pathogenicity test We evaluated 4 fungal isolates in the pathogenicity test: HARP 15-01, HARP 15-02, HARP 15-03, and HARP 15-04. Our preparations of the non-infected H. portoricensis fruit included surface-sterilization with 1% sodium hypochlorite for 1 min, washing twice with sterile distilled water non-infected fruits, blotting dry with a sterile paper tissue, and inoculation using the wound/drop or non-wound/ drop method with modifications (Kanchana-udomkan et al. 2004, Lin et al. 2002). Instead of using a drop of conidia suspension in the wounded or unwounded area, we placed a 2-mm2 disk of mycelium from a 7-d-old colony on the area. We incubated the inoculated fruits at 25 °C and 95% relative humidity in a 12-h light/dark cycle. We conducted the experiment twice on undetached fruits at full maturity. We recorded symptoms 10–15 d after inoculation using an ordinal disease-severity scale with 4 classes: (0 = symptomless, 1 = mild rot damage, 2 = moderate rot damage, and 3 = severe rot damage; Madden et al. 2007). Morphological characterization We recorded colony characteristics, fungal-structure measurements, and growth rate from cultures grown on PDA. The phenotypic appearance of the colony resembled typical Colletotrichum colonies; therefore, we based our identification on the morphological taxonomic description for this genus described by Weir et al. (2012). For each isolate, we measured length and width of 25 conidia and appressoria, calculated the length/width ratio, and noted the shape (Chaky et al. 2001, Weir et al. 2012). We performed a growth-rate assay in PDA by measuring the diameter of the colony every day for 10 d during incubation at 25 °C. Molecular characterization We extracted DNA from mycelium using the Qiagen® DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) according to manufacturer’s instructions. We eluted DNA in TE (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) and adjusted the concentration to 25 ng/μl. We performed DNA amplification by PCR using the specific fungal primers: ITS4/ITS5 (615 pb) (White et al. 1990) and T1/T2 for the β-tubulin gene (O’Donnell and Cigelnik 1997, Weir et al. 2012). We conducted the PCR using the AmpliTaq Gold® PCR Master Mix (25 μL) combined with 5 μL of DNA template, 3 μL of each primer (forward and reverse), and 14 μL of ultra-pure water for a total reaction volume of 50 μL. The PCR cycle consisted of 10 min at 95 °C; 35 cycles of 3 seconds at 96 °C, 3 sec at Primer Tm, and 30 sec at 68 °C; 10 sec at 72 °C; and 4 °C for 1 hr. Primer temperatures were 52 °C and 55 °C for ITS and β-tubulin, respectively. We analyzed PCR products by electrophoresis on 1.2 % agarose gels with 2.5 μL of Gel Red in Tris-acetate-EDTA buffer at 110 V for 1 h. We purified products using the QIAquick® PCR purification kit (catalog. no. 28104; Qiagen Inc.) and submitted them to Macrogen, Inc. (Rockville, MD, USA) for sequencing. Nucleotide sequences were analyzed using Blast nucleotide (NCBI, USA) to confirm pathogen identification. We employed Sequencher® version 5.1 and BioEdit (version 7.2.1) for sequence-quality evaluation and a consensus sequence. We used MUSCLE (Edgar, 2004) and ClustalX (Thompson et al. 1997) for multipleCaribbean Naturalist C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 4 sequence alignment, and Mega 6th Edition (Tamura et al. 2013) was used to build “maximum likelihood” phylogenic trees using the bootstrap method with 1000 replications by the Tamura–Nei model. Concatenated trees were constructed for better molecular identification using genes as 1 super gene (Kubatko and Degnan 2007). Statistical analysis We analyzed data in Infostat statistical software (Student version; National University of Cordova, Cordova, Argentina). We compared the differences among the fungi isolates with Fischer ’s least significant difference (LSD) test at P ≤ 0.05. Results We observed anthracnose fruit symptoms in all surveys only during the ripening stage. The fungus attacks the fruit during the ripening stage, about 3 months after anthesis. Symptoms appeared as brown to black spots on ripe fruits that turned into sunken brown to black lesions over time depending on the prevalent relative humidity at the nursery (Fig. 1A). Acervulus and salmon-colored masses of conidia formed in the lesion 3 weeks after the initial symptoms appeare d (Fig. 1B, C). We obtained 4 fungal isolates (HARP 15-01, HARP 15-02, HARP 15-03, and HARP 15-04) from symptomatic fruit tissue of H. portoricensis (Fig. 2A–D). We noted phenotypic differences between the isolates HARP 15-01, HARP 15-03, and HARP 15-04 compared to HARP 15-02 (Fig. 2A–D). Isolates HARP 15-01, HARP 15-03, and HARP 15-04 on PDA showed a colony of grey aerial mycelia, dense and cottony with white edges and masses of salmon-colored conidia in the center. Isolate HARP 15-02 showed grey, aerial, cottony mycelia at first becoming white with few masses of salmon-colored conidia at the inoculation point (Fig. 2). Based on previous phenotypic morphological descriptions, all isolates fitted under the genus Colletotrichum (Cannon et al. 2012, Weir et al. 2012). Considering the daily growth rate, size, and shape of the conidia, and size and shape of the appressoria, all isolates matched the description of Colletotrichum siamense (Prihastuti, L. Cai, & K.D. Hyde) (Prihastuti et al. 2009, Weir et al. 2012). (Table 1; Figs. 3, 4). Conidia were translucent and oval to cylindrical; the appressoria were brown, clavate, and circular (Fig. 3). None of the isolates showed significant differences (P > 0.05) in growth Figure 1. Anthracnose symptoms on the fruit of Harrisia portoricensis. (A) sunken brown to black lesions, (B) acervulus on infected tissue, and (C) sal mon-colored mass of conidia. Caribbean Naturalist 5 C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 rate according to the Fisher LSD test (Fig. 4). We detected no statistically significant differences between isolates in the size of conidia and appressoria (Table 1). In the pathogenicity tests, all Colletotrichum isolates penetrated the tissue, directly and indirectly causing the same symptoms detected in the surveys; thus, Figure 2. Colletotrichum grown colony on potato dextrose agar (PDA). (A) HARP 15-01, (B) HARP 15-02, (C) HARP 15-03, and (D) HARP 15-04. Table 1. Morphological data of the conidia and appressoria of 4 isolates of Colletotrichum siamense. Dash (-) indicates no appressoria observed. Conidial dimensions (μm) Appressoria dimensions (μm) Isolates Length Width Length Width HARP 15-01 11.81 4.91 8.18 5.45 HARP 15-02 11.97 4.89 7.77 5.21 HARP 15-03 12.31 5.11 8.68 5.73 HARP 15-04 11.40 5.50 - - Caribbean Naturalist C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 6 confirming their pathogenicity on H. portoricensis. Control samples (only PDA) did not exhibit symptoms, whereas the 4 isolates produced greater damage in the Figure 3. Conidia (c) and appressoria (a) of Colletotrichum siamense. (A) HARP 15-01, (B) HARP 15-02, (C) HARP 15-03, and (D) HARP 15-04. Figure 4. Daily growth rate of the 4 isolates of Colletotrichum siamense. Caribbean Naturalist 7 C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 inoculated wounded area than in the non-wounded inoculated area. We observed differences between isolates in the levels of severity of the symptoms they caused (Fig. 5). Samples inoculated with HARP 15-03 and HARP 15-04 isolates showed severe rot damage (level 3), while HARP 15-01 and HARP 15-02 showed moderate rot damage according to with the disease-severity scale used (Fig. 5). The nucleotide sequence of the ITS4/ITS5 (rRNA gene) varied from 600 bp to 660 bp, and the sequence from the β-tubulin gene varied from 695 bp to 780 bp. We could not reliably distinguish any of the isolates from other species of Colletotrichum using the ITS sequences obtained; thus, our identification of the Colletotrichum species was based on the β-tubulin gene sequences. The sequences of the 4 isolates showed more than 97% homology with the reference sequence (JX010391) of C. siamense in the GenBank, thus confirming the identity of the causal agent of fruit rot in H. portoricensis. Discussion Our combined data from morphology characterization, pathogenicity tests, and molecular analysis suggest that the causal agent of the fruit-rot symptoms on H. portoricensis was C. siamense. Even with the differences established by the phenotypic appearance of the colony (conidia and appressoria size) and level of disease severity between isolates, we conclude that all isolates represent the same species. Phenotypic variation of isolates of C. siamense on artificial media is considered broadly linked to the conditions under which the isolates are stored (Weir et al. 2012). The sizes of the fungal structures on our isolates (7–18.3 μm and 3–4.3 μm for conidia and appressoria, respectively) matched those previously reported for C. siamense (4.7–8.3 μm and 3.5–5 μm for conidia and appressoria, respectively; Prihastuti et al. 2009). The daily growth rate (7 mm/d) of all Colletotrichum isolates fitted within the values previously established for C. siamense (6.58–11.5 mm/d; Prihastuti et al. 2009), strongly supporting the taxonomic identification of the isolates. Despite the high uniformity between isolates of C. siamense in morphological characteristics, pathogenicity tests on ripped H. portoricensis fruits revealed variability regarding the extension of the infected surface. Recent studies in the Colletotrichum species complex recognized that many species of this genus remain Figure 5. Pathogenicity tests perfomed in the fruit of Harrisia portoricensis. (A) Control (uninfected fruit), (B) inoculated fruit with HARP 15-01 isolate, (C) inoculated with HARP 15-02 isolate, (D) inoculated with HARP 15-03 isolate, and (E) inoculated with HARP 15- 04 isolate. Caribbean Naturalist C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 8 poorly understood regarding their pathogenicity and host preference (Weir et al. 2012). Even given the variability in disease severity between isolates observed in our study, our results on the affect on the fruits indicate the potential for significant impact of this pathogen on the reproduction of this endangered cactus species. Further studies to link the difference in pathogenicity with a genetic difference could potentially separate populations within this fungal species. Species identification based on their cultural characters and growth rates was robustly supported by the phylogenetic analysis of the β-tubulin gene sequences. In our study, the phylogenic analysis based only on the ITS region was not discriminative enough to distinguish beteween our isolates and species closely related to Colletotrichum siamense, showing ≥98% homology with C. gloesporioides (Penz.) Penz. & Sacc. and C. tropicale E.I. Rojas, S.A. Rehner, & Samuels. The ITS regions can be easily amplified, sequenced, and compared to alternative genes for the broadest range of fungi (Kiss 2012). ITS regions have been proposed as the official fungal DNA barcode markers; however, scientific evidence has shown that these regions do not always discriminate between closely related species (Kiss 2012, Weir et al. 2012). Previous reports on the Colletotrichum species complex established that ITS sequences do not reliably separate C. siamense from C. alienum B. Weir & P.R. Johnst, C. fructicola Prihastuti, L. Cai & K.D. Hyde, or C. tropicale and recommended CAL (Calmodulin) or TUB2 (β-Tubulin 2) primers to best distinguish them (Weir et al. 2012). The partial sequences of the β-tubulin gene showed 99% homology with reference sequences from the GeneBank of C. siamense as well as C. hymenocallidis Yan L. Yang, Zuo Y. Liu, K.D. Hyde & L. Cai for the 4 isolates. Weir et al. (2012), described both C. siamense and C. hymenocallidis within a monophyletic clade that cannot be further subdivided phylogenetically. However, C. siamense and C. hymenocallidis are considered to be synonyms in the Colletotrichum species complex (Weir et al., 2012). Colletotrichum siamense falls within the Musae clade, which includes economically important species such as C. fructicola, C. musae Berk. & M.A. Curtis Arx, Verh., and C. tropicale among others (Weir et al., 2012). The genus Colletotrichum its primarily distributed in tropical and subtropical regions and its known to cause economically important diseases on a wide range of hosts, including woody and herbaceous plants (Cannon et al. 2012). Several species of Colletotrichum associated with tropical crops have been reported in Puerto Rico (Fuentes-Aponte 2015, Rivera-Vargas et al. 2006). Moreover, an additional phylogenetic analysis showed 99% homology of our DNA sequences with reference sequences from the GenBank of C. siamense previously reported associated with Dioscorea rotundata Poir (Yam) in Puerto Rico (data not shown). This finding suggests the possible movement of this fungi from one host to the other in a small geographic area like Puerto Rico. Harrisia portoricensis is an endemic cactus restricted to the small islands of Mona, Monito, and Desecheo in Puerto Rico (USFWS 1990). Since 1913, no populations of H. portoricensis have been identified on the main island; thus, H. portoricensis individuals on the main island are re-introduced or nursery propagated-populations. Caribbean Naturalist 9 C. Soto-Ramos and M. Feliciano-Rivera 2019 No. 56 Although the results shown here are from a nursery-propagated population of H. portoricensis, this pathogen is a potential threat to the conservation of the species and the management strategies established in the recovery plan (USFWS 1996). We have established that C. siamense is very devastating to the fruit of H. portoricensis and could be the cause of the decline in the reproduction of this species on a nursery level. We currently lack information on the associated pest and pathogens to H. portoricensis in their natural habitat. Finally, we suggest additional efforts in Puerto Rico, including surveys in natural habitats to detect the presence of this potential threat to the conservation and propagation of H. portoricensis. Acknowledgments This research was partially supported by USDA NIFA Grant PR.W-2016-03473. We thank Omar Monsegur (Fish and Wildlife Biologist, Endangered Species Program) for kindly providing the specimens of Harrisia portoricensis for this research. We also express our gratitude to agronomists Víctor M. González and Luis Collazo for their technical assistance with the pathogenicity tests and the greenhouses. Literature Cited Agrios, G.N. 2005. Plant Pathology, 5th Edition. Elsevier Academic Press, New York, NY, USA. 922 pp. Cannon, P., U. Damm, P. Johnston, and B. Weir. 2012. Colletotrichum: Current status and future directions. Studies in Mycology 73:181–213. Chaky, J., K. Anderson, M. Moss, and L. Vaillancourt. 2001. Surface hydrophobicity and surface rigidity induce spore germination in Colletotrichum graminicola. Phytopathology, 91(6):558–64. DOI:10.1094/PHYTO.2001.91.6.558. Choi, Y.W., K.D. Hyde, and W.H. Ho. 1999. Single-spore isolation of fungi. Fungal Diversity 3:29–38. Edgar, R.C. 2004. MUSCLE: Multiple-sequence alignment with high accuracy and high throughput. 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