Brown spot is the most important disease for the culture of cactus prickly pear (Nopalea cochenillifera (L) Salm-Dyck; Cactaceae family), and in many countries, Alternaria spp. are commonly associated with the disease. In Brazil, only three species of Alternaria are associated with brown spot: A. alternata, A. tenuissima, and A. longipes. However, other Alternaria spp. may be involved in the etiology of the disease. This study aimed to identify Alternaria species associated with prickly pear brown spot in Northeast Brazil. Cladodes presenting symptoms of brown spot were collected from three counties in the state of Alagoas and three in the state of Pernambuco. Isolation, obtaining monosporic cultures, and pathogenicity tests were performed. For correct taxonomic positioning of the isolates, morphological and molecular analyses were performed using the internal transcribed spacer (ITS) region and the translation elongation factor 1-alpha (tef1), Alternaria major allergen (Alt a1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and second largest subunit of RNA polymerase (rpb2). Fourteen pathogenic isolates were obtained, and according to the morphological and molecular analyses, three species of Alternaria were identified: A. gossypina, A. jacinthicola, and A. tomato, all belonging to the Alternata section. A. gossypina, A. jacinthicola, and A. tomato are described for the first time in Nopalea cochenillifera worldwide.
Brown spot is the main disease of the cactus prickly pear (Nopalea cochenillifera (L) Salm-Dyck; Cactaceae family) in Brazil. It is characterized by brown to black spots, circular in shape, which may coalesce to form large necrotic areas and cause perforations due to the fall of infected tissue (Conforto et al. 2019). Currently, brown spot is predominantly reported in the Northeast region, with greater frequency in the main producing regions in the states of Alagoas and Pernambuco, where cactus prickly pear culture is of high economic importance (Santos et al. 2001; Souza et al. 2010; Lima et al. 2011; Barbosa et al. 2012; Silva et al. 2014).The disease, initially named Alternaria spot, was reported for the first time in the city of São Bento do Una in the state of Pernambuco, Brazil, and was attributed to A. alternata. Since, A. tenuissima and A. longipes have also been identified as causing brown spot (Lima et al. 2011; Conforto et al. 2019).
The Alternaria genus is distributed worldwide, with saprophytic, endophytic, and mainly pathogenic species. It is a group of fungi with great adaptability and survival in different environmental conditions, causing serious problems to a wide range of host plants (Gabriel et al. 2017; Siciliano et al. 2018). The identification of its species is a very controversial issue, initially resolved by the morphology of reproductive structures, colony coloring, and host specificity (Simmons 1967, 1971, 1989, 1992, 2007), however, due to the overlap and high morphological plasticity between species, the use of multilocus phylogenetic analyses, based on different regions (ITS and mtSSU) and protein coding genes (gapdh, Alt a1, tub, tef1, rpb2, Chitin synthase, Calmodulin, ATPase), has become standard for taxonomy within the genus, grouping the species into sections, according to the phylogenetic relationship between them (Lawrence et al. 2013; Lawrence et al. 2015).
Thus, the objective of this study was to identify Alternaria species associated with brown spot of cactus prickly pear (N. cochenillifera) based on morphological and phylogenetic analyses in the Brazilian Northeast region.
Material and methods
This work was conducted in the Molecular Phytopathology Laboratory of the agrarian Engineering and Sciences Campus of the Universidade Federal de Alagoas (UFAL), located in the city of Rio Largo.
Obtaining and preserving isolates
Alternaria isolates were obtained from cladodes with brown spot symptoms, during the rainy season in 2018 and 2019, in the state of Alagoas, in the cities of Cacimbinhas (09°40′16”S/36°99′14”W), Estrela de Alagoas (09°23′124”S/36°45′36”W) and Palmeira dos Índios (09°40′61”S/36°63′31”W), and Pernambuco, in the cities of Bom Conselho (09°10′12”S/36°40′48”W), São João (08°52′33”S/36°22′01”W) and Lajedo (08°65′83”S/36°32′97”W). Per sampled are, 25 cladodes were collected.
In the laboratory, cladodes were washed under running water and dried using sterilized paper towels. Isolations were completed by removing fragments from symptomatic tissue at the interface between the necrotic tissue and non-symptomatic tissue, which were disinfested with 70% alcohol solution for 30 s, sodium hypochlorite (1%) for 1 min, washed twice in sterile distilled water (SDW), and placed to dry on sterile filter paper. After they were dried, the fragments were transferred to Petri dishes containing potato dextrose agar (PDA), then maintained in BOD at 25 ± 1 °C in the dark for 7 days.
After the colonies were formed, 5 mm disks containing pathogen mycelia were transferred to new Petri dishes containing PDA medium, maintained at a temperature of 25 °C until the emergence of the pathogen’s reproductive structures, for later identification and obtaining monosporic cultures. After obtaining the monosporic cultures, isolates were preserved using three methods: test tubes containing PDA culture medium, Castellani, and on autoclaved filter paper (Castellani 1960). The fungal colonies were deposited in the Coleção de Fitopatógenos da Universidade Federal de Alagoas (COUFAL).
Pathogenicity of fourteen isolates were tested on healthy cladodes detached from prickly pears in a plantation located in the county of Rio Largo, Alagoas, Brazil. The cladodes were washed in running water and disinfested by immersion in 1% NaOCl solution for 3 min, rinsed in SDW and dried at room temperature. Two inoculation methods were used. For both methods the cladodes were wounded with the aid of a previously sterilized needle. In first method, the wound was inoculated with a 5 mm PDA disks containing fungal structures. For the control, PDA-only disks. The pathogenicity was also tested using a spore suspension. The inoculations were performed by applying a drop of 30 μL of a suspension with 106 spores mL−1 (quantified in a hemocytometer), on each inoculation spot, and in the control treatment a drop of 30 μL SDW was applied. Each cladode was packaged separately in plastic bags containing cotton moistened with SDW, maintained in a humid environment for 48 h, and then kept in a BOD incubator (Biochemical Oxygen Demand) at 25 ± 1 °C with a 12-h photoperiod for up to 10 days. Three repetitions per isolate were used, where each repetition consisted of a cladode with four wounds, one being the control (Flores-Flores et al. 2013; Feijó et al. 2019).
For molecular characterization, the extraction of DNA was carried out from the mycelial mass of each isolate grown in L-Asparagine liquid medium for five days in a 50 mL Erlenmeyer flask, which was incubated at room temperature (25 ± 1 °C) with a photoperiod of 12 h. The protocol used for extraction was the CTAB method adapted from Doyle and Doyle (1987). Polymerase chain reactions (PCR) for all genes were prepared in a final volume of 30 μl: 10X buffer (3 μL), MgCl2 50 mM (0.9 μL), 10 mM DNTP’s (2.4 μL), 10 μM of each oligonucleotide (2 μL), 1 U Taq DNA polymerase (0.2 μL) and DNA (1 μL, 25 ng / μL). Primer pairs that code for the ITS, Alt a1, tef1, rpb2, and gapdg genomic regions were used (Table 1). The PCR products were then sent for purification and sequencing directly at the Macrogen company in Seoul, South Korea.
The nucleotide sequences obtained were assembled with the Codon Code Aligner v. 6. 0. 2 software and analyzed visually to obtain a consensus sequence of the amplified region for all isolates. The arrangement of nucleotides in ambiguous positions was corrected by comparing the sequences in sense and antisense directions. The initial analyses were performed with the BLASTn algorithm (Altschul et al. 1990) and the non-redundant GenBank database. Reference sequences for several species of the genus were retrieved from GenBank (Table 2) and used for reconstruction of the phylogenetic tree.
Multiple alignments for nucleotide sequences were obtained using the MUSCLE algorithm (Edgar 2004) in the MEGA v. 6 program (Tamura et al. 2013). The phylogenies of Bayesian Inference (BI) for the sequence data of ITS, Alt a1, tef1, rpb2, and gapdh, were built individually in the CIPRES portal (Miller et al. 2010) using MrBayes v. 3.2.3 (Ronquist et al. 2012). The best evolutionary model for the partitioning analyses was performed on the concatenated sequences by PartitionFinder 2.1.1 according to Akaike Information Criterion (AIC) (Lanfear et al. 2016). Analyses were running for 10 million generations using four chains and sampled every 1000 generations, for a total of 10,000 trees. The first 2500 trees were discarded as a burn-in phase. Later probabilities (Rannala and Yang 1996) were determined from a majority-rule consensus tree generated with the remaining 7500 trees. The trees were visualized and edited using the FigTree program v.1.4 (ztree.bio.ed.ac.uk/software/figtree) and Inkscape (https://inkscape.org/pt/).
Morphological and cultural characterization
For cultural characterization, disks of culture medium containing mycelium of the pathogen were removed from the edges of the colonies (grown for 7 days) and transferred to Petri plates containing synthetic PDA. The treatments were kept in a BOD incubator at 25 ± 1 °C and a 12-h photoperiod. The experimental design was completely randomized with five replications per species, each repetition being a petri plate. The coloring of the colonies and aspect of the aerial mycelium were observed.
For morphological characterization, the size and shape of the spores (n = 50), break length, conidiophores, and branching pattern from the isolates were obtained in potato carrot agar (PCA) maintained at a temperature of 25 ± 1 °C for 10 days and photoperiod of 8 h of light and 16 h of darkness (Simmons 2007). The pictures were captured by a digital camera (Olympus IX2-SLP) attached to the optical microscope with 400 x magnification using the Cellsenses Standard software (SAMSUNG SDC-415®) and later used to determine the length and width of the conidia.
Obtaining the isolates
Fourteen fungal isolates were obtained showing morphological characteristics that were compatible with the Alternaria genus, six of which were obtained in the state of Alagoas (COUFAL0300, COUFAL0255, COUFAL0256, COUFAL0257, COUFAL0301, and COUFAL0254), and eight were from Pernambuco (COUFAL0294, COUFAL0295, COUFAL0296, COUFAL0258, COUFAL0297, COUFAL0299, COUFAL0259, and COUFAL0298).
All isolates caused symptoms on the cladode cactus prickly pear, regardless of inoculation method. Symptoms were first observed seven days post-inoculation (dpi) and evaluated at 10 dpi. In inoculations with disks containing pathogen mycelial, the lesions were circular and necrotic, which subsequently advanced to other face of the cladode, and presented an average lesion size of 11.21, 9.97, and 9.35 mm for the species Alternaria gossypina, Alternaria jacinthicola, and Alternaria tomato, respectively. Inoculations with the suspension presented a smaller average lesion size, 7.21 (A. gossypina), 7.08 (A. jacinthicola), and 7.02 mm (A. tomato) for all species compared to the tests performed with disks (Fig. 1). The lesions were also circular and necrotic; however, they were not observed on the other face of the cladode. After the pathogenicity test, re-isolations were performed, thus, confirming Koch’s postulates.
Approximately 606 bases were determined for ITS, 347 bases for tef1, 957 bases for rpb2, 472 bases for Alt a1, and 579 bases for gapdh. Congruence analyses did not reveal conflict between the data sets of the ITS, Alt a1, tef1, rpb2, and gapdh sequences, therefore, they were combined. The generated ML and IB trees showed the same topology, therefore, only the BI tree was presented in this study. The combined data set of the five loci, included 57 taxa with A. alternantherae (CBS 124392) as an outgroup taxon. The alignment presented 2961 characters, of which 345 were parsimony informative sites, 2059 were conserved sites, and 868 were variable sites. The locus limits in the alignments were: 1–597 for rpb2, 958–1304 for tef1, 135–1883 for gapdh, 1884–2489 for ITS, and 2490–2961 for Alt a1. For phylogenetic analyses of the BI, according to AIC the TIM + G model was selected for ITS, TRNEF+G for rpb2 and tef1, HKY + G for Alt a1, and TRN + G for gapdh, and for the combined data, the model GTR + G was used (Study 26,406 deposited in TreeBASE).
The results showed that the isolates of this study were grouped into three distinct clades, where: COUFAL0294, COUFAL0295, COUFAL0296, COUFAL0258, and COUFAL0297 were from Bom Conselho/PE, COUFAL0259 and COUFAL0298 were from Lajedo/PE, COUFAL099 was from Estrela de Alagoas/AL, grouped with other sequences of A. gossypina, with the posterior probability of 1 in the phylogenetic tree; isolates COUFAL0255 and COUFAL0256 were from Estrela de Alagoas/AL and isolate COUFAL0257 was from Palmeira dos Índios/AL, grouped with A. jacinthicola with Bayesian support of 0.98 in the phylogenetic tree; and isolate COUFAL0254 was from Cacimbinhas/AL and COUFAL0301 was from Estrela de Alagoas/AL, grouped with the species A. tomato with a level of Bayesian support 1 in the phylogenetic tree (Fig. 2), all belonging to the same section, Alternata. The species A. gossypina was the most frequently isolated in this study, with most isolates (88.8%) in the state of Pernambuco and only 11.2% obtained in the state of Alagoas. Alternaria jacinthicola, as well as A. tomato, were obtained only in the state of Alagoas, with 100% isolation prevalence.
Morphological and cultural characterization.
Alternaria jacinthicola presented colonies with regular edges, whitish gray aerial mycelium, and on the reverse side, a dark brown color with white edges. The species A. gossypina presented colonies with regular edges, cottony aerial mycelium, with whitish to gray color on the surface, a light brown color on the reverse side, and presence of concentric rings with white borders. The A. tomato species presented colonies with regular edges on the back, cottony aerial mycelium of a whitish gray color, and the reverse side of the colonies showed a dark brown color in the center and a lighter color toward the edges (Fig. 3).
In the morphological analyses, all species had unique conidiophores, in a chain with 2–6 conidia, lateral to the hyphae or terminals, with a dark brown color. Alternaria jacinthicola presented brown conidia, generally in chains of 2–6, rarely solitary, ellipsoid or obclavate, with smooth walls. The conidia measured (15.22) 19.65 (28.00) × (4.33) 6.30 (8.76) μm in size with 3–4 transverse septa and 1 longitudinal septum with a beak from 5 to 15.79 × 2 to 4 μm in size. Alternaria gossypina presented brown conidia, generally in chains of 2–3 conidia, rarely solitary, ellipsoid or obclavate, with smooth walls. The conidia measured (14.27) 21.39 (28.53) × (5.04) 7.51 (10.69) μm in size with 5–7 transverse septa and 1–2 longitudinal septa with a beak from 3.5–14.32 × 2 to 3.9 μm in size. Alternaria tomato presented brown conidia, septate, obclavate, simple or grouped in chains of 2 to 4 conidia measuring (13.5) 19.03 (24.45) × (4.62) 6.85 (9.56) μm in size, with four transverse septa, one longitudinal septum, and the beak measuring 4–8.65 × 1 to 2 μm in size (Fig. 3).
Through the combined analyses of the ITS region and gapdh, Alt a1, tef1, and rpb2 genes associated with morphology, A. gossypina, A. jacinthicola, and A. tomato were identified and characterized in this study, grouping them into three distinct clades, within the Alternata section, as pathogens causing brown spot in the prickly pear (Nopalea cochenillifera).
The identification of Alternaria spp. within the Alternata section, which includes “small conidia” species, has been somewhat challenging, due to the plasticity of morphological characteristics initially used in the taxonomy. Today, the use of molecular techniques for this purpose is recommended (Lawrence et al. 2013). Initially the ITS genomic region was often used in the characterization of fungal species, providing strong support to the described morphological groups, however, its use was debated due to the informativeness level of the region (Hyde et al. 2014). Thus, phylogenetic analyses have become indispensable to elucidate the phylogenetic “knots” within the genus Alternaria, and combinations that include the ITS region and protein codes, such as gapdh, Alt a1, tef1, rpb2, Calmodolin, Chitin synthase, ATPase, and tub, can be used (Pryor and Gilbertson 2000; Hong et al. 2005; Rokas and Carroll 2005; Andrew et al. 2009; Pryor et al. 2009; Lawrence et al. 2012; Lawrence et al. 2013, 2014; Woudenberg et al. 2013; Grum-Grzhimaylo et al. 2016).
Regarding the phylogenetic analyses, it was observed in this study that the ITS region and the tef1 gene were incapable of distinguishing the three identified species, probably due to the conservation and slow rate of molecular evolution of these genomic regions, as described by other authors (Stiller and Hall 1997; Baldauf and Palmer 1993; Cheney et al. 2001; Lawrence et al. 2013). In studies performed by Ding et al. (2019), it was observed that the use of protein coder genes together (gapdh, rpb2, Alt a1, Calmodulin, and ATPase) revealed a more strongly supported Bayesian Inference topology within the Alternata section, and the Alt a1 gene was the most informative. It was also observed that the data of the Alt a1 gene showed better topological resolution among all the genes used here. However, the combination of these genomic regions proved to be superior to the other alternatives derived from a single isolated region in the identification of Alternaria spp. belonging to the Alternata section in this study.
Thus, it is possible to affirm that the need to use combined genes in the precise identification of species that make up the Alternata section, is due to the minimal molecular variation, reported by other authors (Woudenberg et al. 2015) and visualized in this study, that is, few fixed nucleotides can separate one species from another, when the genes are analyzed individually. Alternaria gossypina shows only three fixed nucleotides in its genome, however, it can easily be distinguished from A. alternata using combined molecular data (Woudenberg et al. 2015). This species has a close phylogenetic relationship with A. longipes, recently associated with brown spot in N. cochenillifera in the state of Pernambuco (Conforto et al. 2019). Alternaria tomato and A. jacinthicola, also show a low number of fixed nucleotides, due to their close relationship with A. burnsii, which grouped in a clade called “clade brothers” (Luo et al. 2018).
Among the identified species in this study, A. gossypina was the most prevalent, and the only one of the three species present in both collection states (Alagoas and Pernambuco). This species was first recognized in 1931 in the cotton culture in Zimbabwe, and today, it is responsible for diseases in different hosts in different parts of the world (Farr and Rossman 2019). To date, there are no reports of this species associated to prickly pear in the world, nor reports in other cultures in Brazil.
Alternaria jacinthicola was described as a new species in 2011 in West Africa, and it has been reported in peanuts (Republic of Mauritius), clove (China), and wheat (Oman) (Farr and Rossman 2019). To date, there are no reports of this species in Brazil associated with any culture, therefore, this is the first report of A. jacinthicola in Brazil and in the world, causing prickly pear disease.
Alternaria tomato was originally described in 1939 infecting Solanum lycopersicum. Currently, it has a restricted host range, being reported, in addition to tomatoes, in potatoes (Solanum tuberosum), beans (Phaseolus vulgaris), passion fruit (Passiflora edulis), and more recently, causing leaf spot in sunflower (Helianthus annuus) cultivations in Mexico (Farr and Rossman 2019). Furthermore, this species is known to produce mycotoxins in foods, causing problems in humans (Shinha and Bhatnagar 1998). Thus, this is the first report of this species infecting N. cochenillifera in the world, and the first occurrence of this species in Brazil.
Although there are no reports on the presence of the species A. jacinthicola, A. tomato, and A. gossypina in Brazil in any other culture, it is possible that they occur in the country, not only in N. cochenillifera, and this is probably due to the scarcity of studies focused on multilocus phylogenetic analyses of the Alternaria genus, since morphological analyses alone or in conjunction with analyses of the ITS region are unable to separate Alternaria species, especially within the Alternata section, where the species are considered indistinguishable morphologically.
In prickly pear, Alternaria species have already been reported as causing spots in Opuntia ficus-indica cladodes in Mexico (A. alternata), Egypt (A. alternata), South Africa (A. tenuissima), and Brazil (A. tenuis). In N. cochenillifera, A. tenuissima and, more recently, A. alternata and A. longipes have been reported to be associated with brown spot in Brazil (Farr and Rossman 2019).
In the morphological analyses, the identified species presented characteristics similar to those of previous studies for the species A. gossypina, A. jacinthicola, and A. tomato (Hopkins 1931; Luo et al. 2018; Poudel et al. 2019). Colony colorations varied compared to other studies, and according to Simmons 1992, these variations occur due to the cultivation conditions and culture medium used; therefore, they are not reliable for species delimitation (Lawrence et al. 2016).
Regarding pathogenicity, suspension tests were carried out in the present study for the identified species, however, despite causing symptoms in the cladode cactus prickly pear, they presented smaller average lesion size in comparison to tests with disks containing pathogen structures. According to the literature, tests with spore suspension are scarce for cactus prickly pear culture, regardless of the pathogens in question, probably because they produce small lesions, as observed in this study, confirming that the pathogenicity tests performed with disks containing structures pathogen are more recommended for the culture cactus prickly pear (Flores-Flores et al. 2013; Oliveira et al. 2018; Feijó et al. 2019; Conforto et al. 2019).
Finally, our findings revealed a greater diversity of Alternaria spp. associated with brown spot in prickly pear, where we can include the species identified here A. gossypina, A. jacinthicola, and A. tomato as new etiological agents of the disease. It also provides data that can be useful to identify the best control measures for an adequate management of the disease in prickly pear plantation areas.
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This work was supported by Fundação de Amparo à Pesquisa de Alagoas – FAPEAL. We also thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES for a masters fellowiship.
This research article is not submitted elsewhere for publication and this manuscript complies with the Ethical Rules applicable for this journal.
This article does not contain any studies with human participants or animals performed by any of the authors.
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The authors declare that they have no competing interest.
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Infante, N.B., da Silva, G.C.S., Feijó, F.M. et al. Alternaria species associated with cladode brown spot in cactus prickly pear (Nopalea cochenillifera). Eur J Plant Pathol (2021). https://doi.org/10.1007/s10658-021-02236-5
- Fungal diseases
- Multilocus analyses