Alexandre Reis Machado

First Report of Collar and Root Rot of Physic Nut ( Jatropha curcas ) Caused by Neoscytalidium dimidiatum in Brazil

Physic nut (Jatropha curcas L.; Euphorbiaeae) has become important in Brazil due to its potential as a feedstock for biodiesel production. In October 2010, during routine monitoring of fields in the state of Piauí, several plants were observed with symptoms of collar and root rot. Initially, plants appeared withered and chlorotic, and later became defoliated. Roots and collars of affected plants also appeared rotten with black fungal structures directly observed. Symptomatic tissue fragments of 5 mm diameter were washed with 70% ethanol, 1% sodium hypochlorite, and sterilized water, before being placed in petri dishes containing potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were then obtained by single-spore isolation. The fungus isolated was grown on plates containing 2% water agar overlaid with sterilized corn straw or pine twigs and incubated at 25°C under a photoperiod of 12 h for 4 weeks to induce the formation of fruiting bodies. Thirty measurements of all of the relevant morphological characters were made using a light microscope for the identification of the species. On PDA, isolates initially appeared white and became dark after 7 days. The aerial mycelia formed chains of zero- to one-septate arthroconidia, oblong to globose, initially hyaline that became brown and with a thick wall with age. The dimensions of arthroconidia were 4 to 12 × 2.5 to 8 μm. The formation of pycnidia was observed on the plates with corn straw and pines twigs. These were dark, with a globose base up to 250 μm and a neck up to 810 μm. Conidiogenous cells were holoblastic, lageniform to ampulliform, hyaline, and 6 to 10 × 1.5 to 2.5 μm. Conidia were hyaline, ellipsoid to nearly fusiform, and 8 to 12 × 4 to 5 μm. Septate and dark conidia were not observed. DNA was extracted from one isolate following Wizard Genomic DNA Purification Kit procedures and amplified using primers ITS1 and ITS4. Products were directly sequencing by Macrogen, Korea. The 856-bp sequence obtained was deposited in GenBank (Accession No. JQ927342). The sequence was 99% similar to Neoscytalidium dimidiatum (Penz.) Crous & Slippers, further supporting the identification by morphology. Pathogenicity tests were conducted by using 6 mm disks removed from the outer bark of the collar region of healthy plants using a sterile cork borer, and 6-mm diameter plugs were placed in each wound. Five plants were inoculated with the isolate and five plants inoculated with an isolate-free agar plug. Below these, pieces of moistened cotton were placed and covered with Parafilm. After 60 days, all inoculated plants reproduced the symptoms observed in the field, and the pathogen was successfully reisolated. All non-inoculated plants remained healthy. The genus Neoscytalidium includes species having Scytalidium-like synanamorphs in the aerial mycelia and Fusicoccum-like conidia in the pycnidia (1). Currently, this species is reported to cause diseases in fig, mango, and orange (2,3). To our knowledge, this is the first report of collar and rot root caused by N. dimidiatum in J. curcas and of this fungus in Brazil. It seems likely the disease exists in areas beyond Piauí and could cause important losses for biodiesel production. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) G. Polizzi et al. Plant Dis. 93:1215, 2009. (3) J. D. Ray et al. Austral. Plant Dis. Notes 5:48, 2010.

First Report of Leaf Spot Disease Caused by Cercosporella pfaffiae on Brazilian Ginseng (Pfaffia glomerata) in Brazil

Pfaffia glomerata (Spreng) Pedersen (Amaranthaceae) and other species in this genus, popularly known as Brazilian ginseng, have been marketed and used for many years in folk medicine for the treatment of various diseases (1). In January 2012, samples of P. glomerata with leaf spots were collected in the city of Viçosa, state of Minas Gerais, Brazil. Two samples were deposited in the herbarium at the Universidade Federal de Viçosa (VIC31849 and VIC31851). The diseased leaves were examined using a stereomicroscope (75×). The fungal structures were scraped with a scalpel from the plant surface and mounted in lactophenol. Thirty measurements of all of the relevant morphological characters were obtained using light microscopy for the identification of the species. To confirm the identification, fungal DNA from single-spore pure culture was isolated from the diseased leaves on PDA, and the DNA was amplified using primers ITS1 and ITS4 for the ITS region (GenBank Accession No. JQ990331) and LR0R and LR5 for partial 28S rDNA (Accession No. JQ990330). Sequencing was performed by Macrogen, Korea. The symptoms observed were leaf spots, subcircular, usually up to 6 mm diameter, initially yellowish becoming brown to reddish, margin indefinite, with the formation of fungal structures, hypophyllous, white, scattered, or grouped. Conidiophores were very numerous in dense subsynnematal fascicles, moderately brown at the base but for most of the length subhyaline, 42.5 to 350 × 2.5 to 3.5 μm, showing conidial scars. Conidia formed singly, 22.5 to 77.5 × 5 to 6 μm, hyaline, hilum slightly thickened, and refractive. These characteristics show that the fungus found on P. glomerata matched well with the description of Cercosporella pfaffiae (2). Kochs postulates were fulfilled by inoculation of 6-mm-diameter PDA plugs with the isolate mycelia on leaves of P. glomerata. Six plants were inoculated with the isolate and six plants were inoculated with an isolate-free agar plug. Inoculated plants were maintained in a moist chamber for 24 hours and subsequently in a greenhouse at 26°C. Leaf spot was observed in inoculated plants 15 days after inoculation, and symptoms were similar to those in the field. All non-inoculated plants remained healthy. A Megablast search of the NCBI GenBank nucleotide sequence database using the ITS sequence retrieved C. virgaureae as the closest match [GenBank GU214658; Identity = 458/476 (96%), Gaps = 2/476 (0%)]. To confirm the identification, Bayesian inference analyses were employed, and the tree was deposited in TreeBASE (Study S12680). The analysis placed our isolate in the same clade with the type species of Cercosporella. Molecular studies and morphological characteristics confirm our identification. C. pfaffiae has been previously reported in P. iresinoides (H.B.K.) Spreng. in Trinidad and Gomphrena glomerata L. in Argentina (2). To our knowledge, this is the first report of C. pfaffiae causing disease in P. glomerata in Brazil and it may become a serious problem for some medicinal plant growers, due to the severity of the disease and the lack of chemical products for this pathogen. References: (1) Neto et al. J. Ethnopharmacol. 96:87, 2005. (2) U. Braun. A Monograph of Cercosporella, Ramularia and Allied Genera (Phytopathogenic Hyphomycetes). Eching bei Müchen, IHW-Verlage. Vol. 1, p. 68, 1995.

Mycological Diversity Description I

Here, Quambalaria fabacearum and Neopestalotiopsis brasiliensis are introduced as new species from Brazil, isolated as endophyte from Mimosa tenuiflora and causing post-harvest rot disease on fruits of Psidium guajava, respectively. Diaporthe inconspicua is emended to include a more detailed morphological description. Neopestalotiopsis egyptiaca is reported as new to the Americas and as causing post-harvest rot disease on fruits of Psidium guajava, while Umbelopsis isabellina is reported as endophyte.

First report of Gilbertella persicaria causing soft rot in eggplant fruit in Brazil

In March 2017, two eggplant fruit (Solanum melongena cv. Cica) were collected from field production in Cha-Grande, in the state of Pernambuco, Brazil, with circular, light brown and slightly sunken lesions in the apex, extending to the entire fruit in 3 to 4 days and developing a soft rot with abundant mycelial and sporangia production on the surface. After microscopic examination, a fungus belonging to the genus Gilbertella was identified. The fungus was isolated from soft rot observed in the fruit and pure cultures were obtained by transferring a single spore to potato dextrose agar (PDA). Two isolates were obtained and deposited in the culture collection “Micoteca URM Profa. Maria Auxiliadora Cavalcanti” at the Universidade Federal de Pernambuco (Recife, Brazil), as URM7670 and URM7671. Morphological characteristics include spherical, single sporangia with 57.2 to 96.2 µm diameter, yellow-brown to brown when young turning dark brown or black on maturity, and sporangia split longitudinally into two halves to release the spore and expose a columella obvoid, 23.4 to 65 × 26 to 52 µm. The sporangiospores were mostly globose to ellipsoid, smooth, hyaline, aseptate, and 7.8 to 13 × 5.2 to 10.4 µm. The morphological characteristics evaluated corroborate previously published data for Gilbertella persicaria (Benny 1991). The partial sequences of the internal transcribed spacer (ITS) and 28S (LSU) of the rDNA were obtained for the two isolates and deposited in GenBank (accession nos. MF804516, MF804517, MG573061, and MG573062, respectively). The isolates showed a 99% similarity with ITS and LSU sequences of the type strain G. persicaria CBS 190.32 (NR111692 and JN939197, respectively) according to BLAST search. A combined phylogenetic tree obtained by Bayesian method grouped the isolates with the G. persicaria clade with 100% of posterior probability, confirming the identification. Koch’s postulates were conducted by inoculating five asymptomatic eggplant fruit (cv. Cica) previously disinfested with 0.5% hypochlorite and unwounded or superficially wounded with a sterile needle at two equidistant points. Ten milliliters of a sporangiospore suspension (1 × 10⁵ sporangiospores/ml) obtained from a representative isolate (URM7670) were sprayed on the inoculation sites. Sterile distilled water was used as control. The inoculated fruits were maintained in plastic boxes that contained a portion of moistened cotton wool and were maintained in a moist chamber at ∼25°C for 5 days. Wounded inoculated fruit showed the first symptoms (circular, light brown spots) 48 h after inoculation and evolved into soft rot after 5 days. Hyaline hyphae and light brown to dark sporangia were observed over the surface of the infected fruit. The pathogen was reisolated, fulfilling Koch’s postulates. The control and unwounded fruit remained asymptomatic. This species is usually found in soils and dung, but has already been reported to cause rot in peach fruit (Ginting et al. 1996), dragon fruit (Guo et al. 2012), black plum (Pinho et al. 2014), and papaya (Cruz-Lachica et al. 2016). To our knowledge, G. persicaria represents a new report in the world associated with soft rot in eggplant. This work will certainly contribute to the knowledge of pathogens that affect eggplant culture and, consequently, future studies that involve disease management strategies.

First Report of Curvularia eragrostidis Causing Postharvest Rot on Pineapple in Brazil

Pineapple (Ananas comosus L. Merril.) is the main plant of the Bromeliaceae, cultivated economically for the fruits appealing flavor and a refreshing sugar-acid balance. In 2013, fruits with no initially visible symptoms began to show a postharvest rot after 3 days in a market in the municipality of Viçosa, Minas Gerais, Brazil. The rot can rarely be detected from the outside of the fruit, but a longitudinal section allows observation of extension of the affected area toward the center of the fruit. The symptoms initially appear as a dark brown to black rot on surface of the fruits, which gradually enlarges in size, leading to increased rot and disposal of infected fruits. Until now, this disease occurred sporadically and caused small losses. A fungus was isolated from rot observed in fruits from cultivar Pérola and a single-spore culture was deposited in the culture collection of the Universidade Federal de Viçosa (Accession No. COAD 1588). After 7 days of incubation at 25°C, the strain displayed radial growth and gray-white to black colonies. Microscopic observations revealed brown to light brown conidiophores present singly or in groups. The septate, simple or rarely branched conidiophores are straight or curved, up to 245 μm long and 5 μm wide, and some have a geniculate growth pattern near the apex. The conidia are ellipsoidal or barrel-shaped and 22 to 25 μm long and 10 to 12.5 μm wide. The median septum appears as a black band and the cells at each end of the conidia are pale, whereas the intermediate cells are brown or dark brown. Based on morphological characteristics, the fungus was identified as Curvularia eragrostidis (4). To confirm this identification, DNA was extracted and sequences of the internal transcribed spacer (ITS), 28S and 18S rDNA regions were obtained and deposited in GenBank (Accession Nos. KJ541818 to KJ541820). The sequence of the ITS region exhibited 99% identity over 530 bp with other C. eragrostidis sequence in GenBank (JN943449) and Bayesian inference analysis placed our isolate in the same clade with others C. eragrostidis (study S15670 deposited in TreeBASE). Kochs postulates were conducted by inoculating six fruits of pineapple previously disinfected with 2% sodium hypochlorite and washed in sterile distilled water. For inoculation, the isolate was grown in potato dextrose agar (PDA) for 15 days at 25°C. Six millimeter diameter disks were removed from the surface of fruits with a sterile cork borer and replaced with PDA disks containing mycelia from the margins of the culture. An agar plug was deposited in three control fruits and all fruits were maintained at 25°C in plastic trays. Inoculated fruits showed symptoms 7 days after inoculation that were similar to those initially observed in the infected fruits, while control fruits showed no symptoms. C. eragrostidis is a cosmopolitan pathogen that infects hosts from several botanical families (2,4). In Brazil, this fungus causes leaf spot on A. comosus (3) and also infects Allium sativum, Dioscorea alata, D. cayenensis, Oryza sativa, Sorghum bicolor, Vigna unguiculata, and Zea mays (1). To our knowledge, this is the first report of C. eragrostidis causing postharvest rot disease in pineapple in Brazil. Because invasion of the fungus can occur through minute fractures, fruits should be carefully handled to avoid mechanical damage. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases , 18 February 2014. (2) D. S. Manamgoda et al. Fungal Divers. 51:3, 2011. (3) J. J. Ponte et al. Fitopatologia 10:21, 1975. (4) A. Sivanesan. Mycological Papers 158:113, 1987.

Bayesian analyses of five gene regions reveal a new phylogenetic species of Macrophomina associated with charcoal rot on oilseed crops in Brazil

Macrophomina is a genus belonging to Botryosphaeriaceae that comprises well-known necrotrophic pathogens related to hundreds of plant hosts around the world. Historically, M. phaseolina is the causal agent of charcoal rot in several crops, mainly in tropical and subtropical areas around the world. However, after a recent genetic diversity study using morphological and molecular approaches, which resulted in the epitypification of M. phaseolina, and the description of a new Macrophomina species associated with charcoal rot disease, the hypothesis that other cryptic species could be present under the name M. phaseolina was raised. Previous studies in Brazil revealed a high genetic diversity and different levels of aggressiveness of M. phaseolina isolates associated with charcoal rot in oilseed crops. Thus, the aim of the present study was, through phylogenetic and morphological studies, to determine if isolates of Macrophomina obtained from different oilseed crops represent a single species or distinct taxa. Based on the results obtained, it was possible to identify three different Macrophomina species: M. phaseolina, M. pseudophaseolina and a new phylogenetic species, M. euphorbiicola. This is first report of M. pseudophaseolina in Brazil causing charcoal rot on Arachis hypogaea, Gossypium hirsutum and Ricinus communis and associated with seed decay of Jatropha curcas. In addition, a novel species described in the present study, M. euphorbiicola, is reported as the etiological agent of the charcoal rot on R. communis and Jatropha gossypifolia.

Lasiodiplodia hormozganensis causing basal stem rot on Ricinus communis in Brazil

Ricinus communis plants showing symptoms of root and stem rot were observed in the states of Bahia and Paraíba, Brazil. Based on the morphology and phylogenetic analyses of ITS and TEF-1α combined, the causal agents of the observed symptoms were identified as Lasiodiplodia hormozganensis and L. theobromae, pathogenicity was confirmed by fulfilling Koch’s postulates. To our knowledge, this is the first report from any part of the world of L. hormozganensis causing root and stem rot in R. communis.