Fungi on Commelina Benghalensis from Brazil, with Notes on Potential for Weed Biological Control

Trop. plant pathol. (2018) 43:21–35 DOI 10.1007/s40858-017-0189-6

ORIGINAL ARTICLE

Fungi on Commelina benghalensis from Brazil, with notes on potential for weed biological control

Bruno W. Ferreira1 & Janaina L. Alves1 & Bruno E. C. Miranda1 & Robert W. Barreto1

Received: 6 February 2017 /Accepted: 11 September 2017 /Published online: 4 October 2017 # Sociedade Brasileira de Fitopatologia 2017

Abstract Commelina benghalensis (tropical spiderwort - TS) that such exotic fungi, may offer a valuable resource for man- is an invasive herbaceous plant, native to South and Southeast agement of one of the worst agricultural weeds in Brazil and in Asia and one of the worst agricultural weeds in the tropics. Its the tropical world, and require investigation as biological con- management is notoriously difficult because of its ability to trol agents for introduction in Brazil. regenerate from small fragments and its tolerance to glypho- sate applications. There are no published records of biocontrol Keywords Biological control . Commelinaceae . fungal attempts against TS involving either microbial or arthropod pathogens . taxonomy natural enemies. Prior to investigating classical biocontrol agents, surveys have been conducted in Brazil and, more recently, concentrated in Viçosa (state of Minas Gerais) to de- Introduction termine whether fungal pathogens of TS are already present. Five different fungal pathogens were collected. These fungi Commelina benghalensis (Commelinaceae) is an herbaceous were identified as causing the following diseases: Athelia perennial plant native to South and Southeast Asia. It is rolfsii – crown rot, Cercospora cf. sigesbeckiae – leaf spots, known by several common names, among them tropical spi- Corynespora cassiicola – leaf spots, Neopyricularia obtusa derwort (TS), wandering Jew, and trapoeraba in Brazil. It is sp. nov. – leaf spots and Rhizoctonia solani – blight. One of widely distributed in tropical and subtropical regions of the the fungi found on TS represents a new taxon, and the others world (Kissmann 1991). TS reproduces both by true seeds and represent either a new host association, a new geographic by vegetative propagation. When its stems are cut into pieces, record or both, except for Corynespora cassiicola,which they rapidly regenerate giving rise to new plants, facilitating has already been recorded as able to attack TS (ex-tomato the dissemination and perpetuation of the weed in infested isolates, under controlled conditions) in Brazil. The areas (Holm et al. 1977). TS is regarded as one of the world’s mycobiota identified in Brazil is limited and lacks many of worst weeds, affecting 25 crops in 29 countries (Holm et al. the TS-specific fungal pathogens recorded in Africa and Asia, 1977; Webster et al. 2005). In Brazil, it has been reported namely: Kordyana celebensis, Kordyana indica, Phakopsora causing serious problems in soybean and coffee plants. tecta, Puccinia commelinae, Uredo ochracea, Bauerago Infestations of soybean fields lead to reduced production of combensis and Bauerago commelinae. It is conjectured here, grain, difficulties in harvest operations, and depreciation of the grain rating, due to increased moisture content (Kissmann 1991). A single TS plant can produce up to 1600 seeds, which along with its rapid reproductive rate, may cause the formation Section Editor: Alan R. Wood of dense and uniform stands that completely smother other * Robert W. Barreto plants, especially young or small sized species (Holm et al. [email protected] 1977). TS has a membranous sheath enveloping its axillary buds, which prevents the direct contact of these regenerative 1 Departamento de Fitopatologia, Universidade Federal de Viçosa, tissues with herbicides, hindering their absorption and making Viçosa, Minas Gerais 36570-900, Brazil chemical control less efficient. TS also produces seeds from 22 Trop. plant pathol. (2018) 43:21–35 cleistogamous hypogeous flowers formed from the rhizomes in Brazil. Specimens of Phoma sp. and Curvularia sp. were which are slightly larger than the seeds produced by aerial never collected again after the survey in the state of Rio de flowers and which are able to germinate and emerge from Janeiro, suggesting that they may represent just occasional up to 12 cm underground, diminishing the efficiency of pre- occurrence of opportunists or saprophytes. Unfortunately, emergence herbicide applications (Kissmann 1991). the work was discontinued without achieving a complete iden- Strangely, although Ellison and Barreto (2004)considered tification of fungi collected in the surveys and the specimens TS as a potential candidate to become a biocontrol target in were lost. More recently, during a local survey in the munic- Latin America, and Evans (1987) considered some pathogenic ipality of Viçosa, in the state of Minas Gerais, Brazil, it was fungi known to attack TS as having good potential for use as found that most of the fungi collected in previous years were classical biocontrol agents, there are still no published records present and available for a more detailed examination. Here, of attempts to target TS for biological control with either ar- the identity of the collected fungi is reported and a comparison thropod or microbial natural enemies. Nevertheless, a classical of the mycobiota associated with C. benghalensis in Brazil biological control program is being conducted against another and the Old World is provided, with comments on their po- member of the family Commelinaceae during recent years, tential use in weed biocontrol in the future. namely Tradescantia fluminensis, a neotropical species that became a noxious environmental weed in New Zealand and Australia after being introduced as an ornamental plant. Materials and methods Several arthropods species have already been introduced against T. fluminensis in New Zealand (Fowler et al. 2013). A provisional list of fungi collected from TS in the state of Rio Also, the fungus Kordyana brasiliensis, recently described by de Janeiro, along with many other weedy hosts has been pub- Macedo et al. (2016), is being evaluated for its release in lished (Barreto and Evans 1995a), but numerous ad hoc col- Australia and New Zealand (Morin 2015). The use of natural lections were also performed since those early days. The sam- enemies collected in the native range of TS in the Old World ples included in this study were collected in the campus of the (classical biological control) is an attractive alternative for bio- Universidade Federal de Viçosa (Viçosa, state of Minas logical control, providing they are damaging to, co-evolved with, Gerais, Brazil) and its vicinities, in crop and pasture areas as and host-specific to TS, and have remained restricted to those well as in ruderal situations such as roadsides, abandoned areas. Natural enemies with such characteristics could be intro- areas, and gardens where TS occurred. They were dried in a duced into regions where TS became a noxious weed. This ap- plant press and later transferred to paper envelopes and depos- proach has a long history, with many examples of success and an ited in the herbarium of the Universidade Federal de Viçosa excellent safety record (Barton 2004). (Herbarium VIC). The samples were carefully examined un- Concepts and examples of the application of pathogens as der a stereoscopic microscope and fungal structures that ap- weed biocontrol agents have been recently reviewed by peared associated with disease symptoms were isolated with a Barreto et al. (2012). Among the first steps of classical bio- sterile fine pointed needle and, whenever possible, directly control programs against weeds, is surveying the area where transferred onto plates containing vegetable broth-agar the weed problem occurs in order to “avoid a later, superfluous (VBA), as described by Pereira et al. (2003). Pure cultures (and also wasteful and potentially embarrassing) introduction were preserved in potato carrot-agar (PCA) tubes, in silica- of natural enemies that may be already present, but were inef- gel, as described in Dhingra and Sinclair (1996)orin fective and, hence, remained unnoticed in the target area” cryotubes containing 10% glycerol stored at -80 °C. Pure cul- (Barreto et al. 2012). TS was included as a target weed in a tures were deposited in the culture collection of the survey performed in the late 1980s, concentrated in the state of Departamento de Fitopatologia of the Universidade Federal Rio de Janeiro (Barreto 1991). Numerous samples of diseased de Viçosa (COAD). TS plants were collected at many sites, but only a preliminary Observations of the morphology of the fungi were examination of the material was possible at the time, and only made on slides containing free hand sections or fungal four fungi were recorded as associated with diseased tissue material scraped from the diseased tissues mounted in (Barreto and Evans 1995a). At the time, those fungi were lactoglycerol or lactofuchsin. Measurements and illustra- identified at the genus level only, and belonged to Bipolaris tions (photomicrographs) were prepared with an sp. (later identified as Corynespora), Cercospora sp., Olympus BX 51 light microscope fitted with a drawing tube Curvularia sp., and Phoma sp. Since then, numerous later and an Olympus E330 camera. Culture descriptions were based ad hoc collections in other Brazilian states were conducted, on the observation of 11-day-old colonies formed in plates con- but the list of fungi associated with diseased TS tissue did not taining either potato dextrose-agar (PDA) or PCA maintained at increase. In fact, the results of these surveys showed that 25 °C under a 12-h daily/light regime (light provided by two Corynespora was the most common fungal pathogen of TS white and one near-UV lamps placed 35 cm above the plates) or Trop. plant pathol. (2018) 43:21–35 23 in the dark (plates wrapped in aluminum foil). The color termi- In culture: Fast growing colonies (90 mm diam after 4 days) nology follows Rayner (1970). composed of white aerial mycelium, sparse and felty, sclerotia Genomic DNAwas extracted from each culture by growing formed on the surface of the colonies after 15 days; not the fungus on PDA plates for 10 days at 24 °C and removing sporulating. the mycelium from each plate. The FastDNA kit (BIO 101, Material examined: Brazil, state of Minas Gerais, Viçosa, Carlsbad, CA) was used, according to the manufacturer’sin- campus of the Universidade Federal de Viçosa, on living structions. The primers ITS4 and ITS5 (White et al. 1990) stems of Commelina benghalensis, 25th May 2016, B. W. were used to amplify the ITS region as well as the 5.8S Ferreira (VIC 44138, culture COAD 2102). rRNA gene, for Cercospora, Neopyricularia, Corynespora, Notes: Disease symptoms and morphology of the fungus Rhizoctonia and Athelia rolfsii. Part of the translation elonga- indicated readily the fungus involved to be A. rolfsii. It did fit tion factor (TEF) gene of the Cercospora and Neopyricularia well within the description of the fungus provided by Mordue isolates was amplified using the primers EF728F and EF986R (1974). The ITS sequences obtained for COAD 2102 were of (Carbone and Kohn 1999), and part of the calmodulin (CAL) poor quality, even after repeated extraction and sequencing gene was amplified with the primers CAL228F and attempts. Nevertheless, in a BLAST search in GenBank such CAL737R (Carbone and Kohn 1999) for the Cercospora iso- sequences were closest to ITS sequences of A. rolfsii. late. The primers LSU1 and LR5 (Vilgalys and Hester 1990) There are only four published records of A. rolfsii attacking were used to amplify part of the ribosomal large subunit for members of the Commelinacea worldwide, namely: on Neopyricularia. The nucleotide sequence data were obtained Commelina benghalensis inTonga(Dingleyetal.1981), on by DNA sequencing (Macrogen Inc., Korea) employing the Dichorisandra sp. in Florida (Farr and Rossman 2016), on same primers used for PCR amplification. The resulting se- Zebrina pendula in Hawaii (Farr and Rossman 2016)andon quences were aligned with other sequences retrieved from Tradescantia fluminensis in Brazil (Macedo et al. 2016). GenBank, including those of selected outgroups, using the Athelia rolfsii is a generalist plant pathogen known to be an MEGA 5.0 software and, when necessary, manually adjusted . important pathogen of various crops (Harlton et al. 1995)and, Bayesian inference analyses were conducted and the best-fit thus, is of no interest for biocontrol. This is the first record of evolutionary model was determined by comparing different evo- A. rolfsii on C. benghalensis in Brazil. lutionary models via the Akaike information criterion using PAUP (version 4.0b10, Sinauer Associates) and MrModeltest Cercospora cf. sigesbeckiae Katsuki, Bull. Agric. 2.2 (Nylander 2004). The Markov chain Monte Carlo Improv. Sect. Econ. Dept. Fukuoka Pref. 1: 22.1949 (MCMC) analysis used four chains and were run for 1,000,000 (Figs. 1a, 2a, b and c). generations. Trees were saved every 1000 generations. The first GenBank: ITS sequence KY351634; TEF sequence 25% of sampled trees were discarded as burn-in. The analysis KY287251; CAL sequence KY287250. was hosted by CIPRES science gateway portal at San Diego Lesions on living leaves, starting as necrotic dots, becom- supercomputer center (Miller et al. 2010). Phylogenetic trees ing sub-circular to irregular, 4–6 mm diam, light brown, often were visualized with the program FigTree v1.3.1 (Rambaut with grayish centre with dark brown margin, becoming 2009). Collection details and GenBank accession numbers of crowded and coalescing along the leaf margins and leading isolates included in this study are provided in Table 1. to leaf blight. Internal mycelium indistinct. External mycelium absent. Stromata limited to few angular cells, 11–37 × 13– 42 μm. Conidiophores amphigenous, fasciculate (4–14 conid- Results iophores per fascicle), cylindrical, 80–177 × 4–5 μm, un- branched, straight to curved, upper third geniculate, 2–6sep- Athelia rolfsii (Curzi)C.C.Tu&Kimbr.,Botanical tate, medium to dark brown, smooth. Conidiogenous cells Gazette Crawfordsville 139: 460.1978. (Figs. 1dand2h). integrated, holoblastic, subcylindrical, terminal, sympodial, GenBank: ITS sequence KY351638 21–48 × 2–4 μm, pale brown. Conidiogenous loci 1–5per Discoloration of tissue starting on the main stem leading to cell, 3–4 μm diam, strongly thickened, darkened. Conidia soft crown rot and plant death, accompanied by formation of a obclavate to filiform, 57–335 × 2–4 μm, straight to slightly fan of white mycelial mat over diseased tissues and spreading curved, with acute apex and obtuse base; indistinctly over soil and organic debris. Sclerotia formed on mycelium, multiseptate, hilum darkened and thickened, hyaline, smooth. round, white when young, becoming brown to dark brown In culture: Slow-growing (2.8–3 cm diam after 28 days), with age, subspherical to spherical, 0.8–2 mm. Primary my- flat with entire edges, aerial mycelium cottonose, pale oliva- celium, 4–7 μm diam, septate, with clamp connections, sec- ceous grey to mouse gray centrally with lilac periphery, ondary mycelium, 2 μm diam, hyaline and smooth. For a reverse violet slate with purple edge, with diurnal zona- complete description, see Mordue (1974). tion; not sporulating. 24 Table 1 Collection details and GenBank accession numbers of isolates included in this study

Species Culture accession numbers Host/isolation source Country Gen Bank accession numbers

ITS TEF CAL LSU

Barretomyces calatheae CBMAI 1060 (ex-type) Calathea longifolia Brazil KM484831 ――KM485045 Corynespora cassiicola AS49 Solanum lycopersicum American Samoa FJ852574 ――― AS54 Vigna unguiculata American Samoa FJ852576 ――― AS67 Commelina benghalensis American Samoa FJ852579 ――― Doa 16b Carica papaya Brazil FJ852588 ――― BZ JMP216a Lantana camara Brazil FJ852589 ――― BZ JMP217 Solanum lycopersicum Brazil FJ852590 ――― BZ JMP218 Glycine max Brazil FJ852591 ――― BZ RWB321 Plectranthus barbatus Brazil FJ852592 ――― Corynespora citricola CBS169.77 – FJ852594 ――― Cercospora apii CBS 114418; CPC 10924 Apium graveolens Italy AY840517 AY840484 AY840415 ― CBS 121.31; CPC 5073 Beta vulgaris Austria AY343371 AY343334 AY840411 ― CBS 132683; CPC 16663 Moluccella laevis Zimbabwe JX143531 JX143285 JX142793 ― MUCC 593 Apium graveolens Japan JX143536 JX143290 JX142798 ― Cercospora kikuchii CBS 128.27; CPC 5068 (Type) Glycine max Japan DQ835070 DQ835088 DQ835134 ― CBS 132633; CPC 16578 Glycine max Argentina JX143619 JX143378 JX142886 ― CBS 135.28; CPC 5067 Glycine max Japan DQ835071 DQ835089 DQ835135 ― Cercospora cf. sigesbeckiae sigesbeckiae CBS 132601; CPC 10664 Sigesbeckia glabrescens South Korea JX143650 JX143409 JX142917 ― MUCC 589; MAFF 305039 Glycine max Japan JX143657 JX143416 JX142924 ― CBS 132675; CPC 14726 Malva verticillata South Korea JX143655 JX143414 JX142922 ― CBS 132606; CPC 10740 Paulownia coreana South Korea JX143651 JX143410 JX142918 ― CBS 132621; CPC 14489 Sigesbeckia pubescens South Korea JX143652 JX143411 JX142919 ― Cercospora zebrina CBS 108.22; CPC 5091 Medicago arabica maculata) – JX143744 JX143503 JX143011 ― CBS 114359; CPC 10901 Hebe sp. New Zealand JX143746 JX143508 JX143016 ― ― CBS 118789; WAC 5106 Trifolium subterraneum Australia JX143747 JX143509 JX143017 43:21 (2018) pathol. plant Trop. CBS 112723; CPC 3957 Trifolium repens Canada AY260079 JX143504 JX143012 ― Septoria provencialis CBS 118910; CPC 12226 Eucalyptus sp. France DQ303096 JX143522 JX143030 ― Macgarvieomyces borealis CBS 461.65 (ex-type) Juncus effusus UK KM484854 ――DQ341511 Macgarvieomyces juncicola CBS 610.82 Juncus effusus Netherlands KM484855 ――KM484970 Magnaporthiopsis maydis CBS 662.82A Zea mays Egypt KM484856 ――KM484971 Neopyricularia commelinicola CBS 128303 = KACC 44637 Commelina communis, South Korea KM484868 ――KM484982 CBS 128306 = KACC 43869 Commelina communis South Korea FJ850123 ――KM484983 CBS 128307 = KACC 44083 Commelina communis South Korea FJ850125 ――KM484984 – CBS 128308 = KACC 43081 (ex-type) Commelina communis, South Korea FJ850122 ――KM484985 35 rp ln ahl 21)43:21 (2018) pathol. plant Trop. Table 1 (continued)

Species Culture accession numbers Host/isolation source Country Gen Bank accession numbers

ITS TEF CAL LSU

Pseudopyricularia cyperi CBS 133595 = MAFF 240229 = Cyperus iria Japan KM484872 ――KM484990 HYCI201–1-1(Ci-1 J) (ex-type) Pseudopyricularia higginsii CBS 121934 = 09/2007/1470 Typha orientalis New Zealand KM484875 ――KM484991 Pyricularia grisea BR0029 Digitaria sanguinalis Brazil KM484880 ――KM484995 – Pyricularia oryzae BF0028 Paspalum sp. Burkina Faso KM484886 ――KM484998 35 Pyricularia zingibericola RN0001 Zingiber officinale Réunion KM484941 ――KM485037 Pyricularia pennisetigena Br36 Cenchrus echinatus Brazil KM484933 ――KM485033 Proxipyricularia zingiberis CBS 132355 = MAFF 240221 = Zingiber mioga Japan AB274433 ――KM484987 HYZiM101–1–1-1(Z-1 J) Rhizoctonia solani AG-1, IA A-10 Oryza sativa Japan AB000010 ――― AG-1, IB SFBV-1 Beta vulgaris Japan AB000038 ――― AG-1, IC RH-28 A Beta vulgaris Japan AB000035 ――― AG-2-1 P-2 Solanum tuberosum U.S.A AB000026 ――― AG-2-2, IIIB B60 ― Japan AB000013 ――― AG-2-2, IV BC-10 Beta vulgaris Japan AB000014 ――― AG-3 1600 Nicotiana tabacum U.S.A AB000004 ――― AG-4, HG-I GM-3 Glycine max Japan AB000018 ――― AG-4, HG-II RR5–2 Beta vulgaris Japan AB000036 ――― AG-6, HG-I HAM1–1 Soil Japan AB000019 ――― AG-7 1556 Soil Japan AB000003 ――― AG-8 A68 Triticum sp. Australia AB000011 ――― AG-9, TP V12 M Solanum tuberosum U.S.A AB000046 ――― 25 26 Trop. plant pathol. (2018) 43:21–35

Fig. 1 Commelina benghalensis bearing disease symptoms caused by pathogenic fungi collected in Viçosa (state of Minas Gerais, Brazil): a. Leaf spots caused by Cercospora cf. sigesbeckiae. b. Leaf spots and foliage blight caused by Corynespora cassiicola. c.Blightcausedby Rhizoctonia solani. d.Crownrot caused by Athelia rolfisii. e-f. Leaf spots and foliage blight caused by Neopyricularia obtusa sp. nov

Fig. 2 Fungi collected on Commelina benghalensis in Viçosa (state of Minas Gerais, Brazil): a. Conidia of Cercospora cf. sigesbeckiae (Bar = 20 μm). b. Conidiophores fascicle of C. cf. sigesbeckiae (Bar = 20 μm). c. Conidiogenous cell and loci of C. cf. sigesbeckiae (Bar = 10 μm). d. Conidia of Corynespora cassiicola (Bar = 40 μm). e. Conidiophores of C. cassiicola (Bar = 80 μm). f. Detail of conidiogenous cells of C. cassiicola with attached immature conidium (Bar = 10 μm). g. Hypha of Rhizoctonia solani (Bar = 10 μm). h. Clamp connection on Hypha of Athelia rolfisii (Bar = 10 μm). i. Conidia of Neopyricularia obtusa sp. nov. (Bar = 20 μm). j. Detail of conidiogenous cell of N. obtusa bearing denticles (Bar = 10 μm) Trop. plant pathol. (2018) 43:21–35 27

Material examined: Brazil, campus of the Universidade viable representative isolate of this collection remains for Federal de Viçosa, state of Minas Gerais, Viçosa, on living DNA comparison and no DNA sequences of leaves of Commelina benghalensis, 15th Dec 2012, B. E. C. C. commelinicola are available in public databases for com- Miranda (VIC 39069, culture COAD 2029). parison. It is possible that C. commelinicola and C. cf. Notes: Five Cercospora species have been reported in as- sigesbeckiae will be shown to be synonyms in the future. sociation with members of the Commelinaceae, namely: This is the first report of C. cf. sigesbeckiae associated with Cercospora nudiflorae, Cercospora commelinicola, C. benghalensis in Brazil and worldwide. Cercospora commelinae-salicifoliae, Cercospora polliae- Corynespora cassiicola (Berkeley & M. A. Curtis) C. T. japonicae and Cercospora apii s.lat. Two other species of Wei. Mycological Papers 34:5.1950 (Figs. 1band2d, e, f). Cercospora had previously been recorded on Commelina GenBank: ITS sequence KY351635 benghalensis: Cercospora commelinae and Cercospora Lesions on living leaves circular to irregular, 1–20 mm, benghalensis. However, after examining the type species of dark brown, often target-like with concentric rings, coalescing C. commelinae,Chupp(1954) concluded that this was in fact and leading to severe blight of individual leaves; also causing a misidentified member of Septoria. Crous and Braun (2003) irregular dark brown necrosis of stems. Internal mycelium, recognized C. benghalensis, as a synonym of C. apii. The intercellular, 3–4 μm diam, branched, septate, hyaline to fungus collected in Brazil on TS differs from those already subhyaline. Conidiophores amphigenous, mostly solitary, cy- reported by having longer conidia. Additional morphological lindrical, straight, 340–1720 × 4–7 μm, 6–8 septate, dark data of Cercospora species on Commelinaceae are provided in Table 2. brown, smooth. Conidiogenous cells terminal, integrated, cy- – – μ Phylogeny: BLAST searches resulted respectively in 100% lindrical, proliferating percurrently, 15 137 × 4 8 m, pale – μ identity with C. cf. sigesbeckiae CBS132641 (GenBank brown, smooth. Conidiogenous loci conspicuous, 1.5 5.5 m JX143412), for the TEF region, and 100% identity with C. diam, thickened, darkened. Conidia, either isolate or forming – – cf. sigesbeckiae PP 2012 003 (GenBank KX443802), for the acropetal chains, fusiform to subcylindrical, 170 350 × 8 CAL region, thus, sequences of this taxon were included in 10 μm, base truncate, apex rounded to obtuse, 8–26 phylogenetic studies. Sequences obtained for the TS isolate distoseptate, hilum thickened and darkened, brown to pale were aligned with sequences of Cercospora and Septoria brown, smooth. provencialis as the outgroup retrieved from the GenBank. In culture: Moderate-growing (7 cm diam after 10 days), The phylogenetic relationships of the Cercospora isolate flat with entire edges, dense cottonose or sparse to felty, pale found on TS with the other fungi were inferred by Bayesian olivaceous gray becoming grey olivaceous or white al- analysis of the ITS, TEF and CAL sequences. The final align- ternating with grey olivaceous rings, diurnal zonation ment contained 18 taxa, including the outgroup. In the present or absent; olivaceous black in reverse. Not phylogenetic tree, the fungus from TS grouped in a C. sporulating. cf. sigesbeckiae clade (Fig. 3). A comparison of the Material examined: Brazil, state of Minas Gerais, Viçosa, fungus on TS with the previously described morphology campus of the Universidade Federal de Viçosa, on living for C. cf. sigesbeckiae by Katsuki (1949) showed these leaves of Commelina benghalensis, 22nd July 2013, B. E. C. taxa to be highly similar, confirming the results of the Miranda (VIC 39731, culture COAD1334). molecular analysis. According to Groenewald et al. (2013), Notes: Morphology of the fungus on TS is consistent the application of the name C. cf. sigesbeckiae (based on type with that is described for Corynespora cassiicola in the material from Japan) to this clade can only be tentative since literature (Ellis and Holliday 1971). The identity of the Japanese cultures and sequences are needed for confirmation isolate was confirmed by phylogenetic analysis (Fig. 4). of its identity. Corynespora cassiicola is a member of the Pleosporales, Cercospora cf. sigesbeckiae is a widely distributed polyph- known only as an asexual morph. It is regarded as a spe- agous fungus, attacking hosts belonging to several plant fam- cies complex and there is ongoing work aimed at ilies, including Begoniaceae, Dioscoreaceae, Fabaceae, unraveling its phylogeny, which may better resolve its sta- Malvaceae, Polygonaceae, Urticaceae, Asteraceae, among tus in the future (Macedo et al. 2013). Corynespora others (Farr and Rossman 2016). There is one earlier record cassiicola is, by far, the most common pathogen found of a member of Cercospora on TS from Viçosa (Lustosa and on TS in Brazil, as observed during the last three decades. Barreto 2001). At the time, the fungus was identified as It is a cosmopolitan species, which has been reported caus- C. commelinicola based on morphology. Nevertheless, the ing diseases on leaves, fruits, roots and branches of more morphology described by Lustosa and Barreto (2001)israther than 300 hosts (Farr and Rossman 2016). Among its hosts similar to that of the fungus described above as C. cf. are included important crops such as tomato (Mohanty and sigesbeckiae and it is likely that the fungus was originally Mohanty 1955), soybean (Seaman and Shoermaker 1964), misidentified by Lustosa and Barreto. Unfortunately, no cucumber (Blazquez 1967) and cotton (Jones 1961). 28

Table 2 Morphological data of Cercospora spp. reported on members of the Commelinaceae or of relevance for comparison with Cercospora sp. collected on Commelina benghalensis in Brazil

Species Stromata Caespituli Conidiophores Conidia References

Cercospora apii Substomatal or amphigenous 12.5–160 × 5–8 μm cylindro-obclavate when shorter, longer conidia usually intraepidermal, up to acicular, straight to slightly curved, 3–10-septate, apex 32 μmdiam subacute to obtuse, truncate to obconically truncate and thickened at the base, 35–120 × 3.5–5 μm. Cercospora Stromata substomatal, amphigenous 10–45 × 2–5.5 μm, narrowly obclavate cylindrical, straight to slightly Kar and Mandal (1973) commelinae-salicifoliae subglobose to 0–4(−7)-septate curved, 1–6-septate, apex obtuse to acute, base somewhat irregular, subtruncate to short obconically truncate, 10–40 μmdiam 20–70 × 1.5–3.5 μm. Cercospora commelinicola Stromata 10–45 μm usually epiphyllous (10–)30–100(−155) × 4–- narrowly obclavate-subcylindrical to acicular, Braun and Sivapalan (1999) diam, substomatal or 7 μm, 1- to pluriseptate pluriseptate, apex subacute, base short to long intraepidermal obconically truncate, 40–150(− 200) ×(2–)3–5.5(−6) μm. Cercospora nudiflorae Stromata substomatal, amphigenous (10–)15–50(−80) × 3–6 μ- narrowly obclavate to acicular, indistinctly pluriseptate, Chupp (1954) small, 10–30 μm m, usually 1–3-septate apex acute or subobtuse, base truncate to obconically diam truncate, 20–80 × 1.5–3.5 μm. Cercospora polliae-japonicae Stromata lacking or amphigenous 25–190 × 4–6.5 μm, acicular to somewhat obclavate, straight to somewhat Guo and Jiang (2000) small, 10–45 μm 0–7-septate curved, pluriseptate, apex pointed, base truncate to diam slightly obconically truncate, 40–200 × 2–5 μm. Cercospora cf. sigesbeckiae – hypophyllous 91–140 × 4–5 μm, 3–4 acicular, straight to mildly curved, 5–11 septate, tip Katsuki (1949) septate slightly narrowed to acute, base truncate, 50–300 3–4×3–4 μm. Cercospora cf. sigesbeckiae Stromata reduced to few amphigenous 80–177 × 4–5 μm, 2–6 obclavate to filiform, 57–335 × 2–4 μm, straight to This publication angular cells, septate slightly curved, with acute apex and obtuse base; 11–37 × 13–42 μm indistinctly multiseptate. 43:21 (2018) pathol. plant Trop. – 35 Trop. plant pathol. (2018) 43:21–35 29

Fig. 3 Phylogenetic study of Cercospora cf. sigesbeckiae - multilocus tree inferred from Bayesian analysis based on the combined sequences of the ITS, TEF and CAL regions. Bayesian posterior probabilities are indicated above the nodes. Septoria provencialis represents the outgroup taxon. The isolate obtained from Commelina benghalensis is in bold

Although this fungus is often regarded as polyphagous, Neopyricularia obtusa B. W. Ferreira, J. L. Alves & R. W. physiological specialization of strains has been document- Barreto sp. nov. (Figs. 1e, f, 2i and j). ed by several authors (Spencer and Walter 1969;Pereira MycoBank: MB 821525 et al. 2003; Macedo et al. 2013). Several such groups of GenBank: ITS sequence KY351636; LSU sequence C. cassiicola have been recognized as formae speciales KX925218. after host-specificity was demonstrated. Corynespora Etymology: In reference to the shape of the conidial apex. cassiicola had been previously recorded in Brazil in pre- Leaf spots amphigenous, scattered, circular to subcircular, liminary surveys, but it was mistakenly identified by 6–10 mm diam, pale brownish centrally to grayish with Morandi and Barreto (1995)asBipolaris sp. Such a mis- brownish to dark brown margin, often surrounded by a yellow take is corrected here. Cutrim and Silva (2003)foundthat halo, coalescing with age and leading to whole leaf blight. C. benghalensis was susceptible when inoculated with Internal mycelium indistinct. External mycelium absent. C. cassiicola isolates obtained from tomato in greenhouse Conidiophores hypophyllous, isolate, subcylindrical, inflated experiments and concluded that TS may be an important at the base (6–10 μm), narrowing to 5–9 μm and tappering source of inoculum for target spot disease of tomato. In towards the zig-zagged fertile apex, 112–337.5 × 3–10 μm, 2– their publication Cutrim and Silva (2003) recommended 4 septate, hyaline. Conidiogenous cells terminal, cylindrical, control of TS in tomato fields as a measure to prevent strongly sympodial, denticulate (denticles 1–2×1–2 μm), 66– target spot disease. Such results would appear discourag- 100 × 3–4 μm, hyaline. Conidia solitary, pyriform to ing for the use of C. cassiicola as a biocontrol agent, un- obclavate, 20–30 × 9–11 μm, base rounded tapering progres- less TS-specific isolates are found. This is the first record sively towards obtuse apex, 2-septate, hilum short-stalked (1– of C. cassiicola occurring under natural conditions on TS 2×1–2 μm), hyaline, smooth. in Brazil. There is only one other previous published re- In culture: Slow-growing (6 cm diam after 11 days), flat cord of this plant-fungus association, and it is from Samoa with entire edges, aerial mycelium cottony, white to pale (Dixon et al. 2009). straw, sparse centrally; not sporulating. 30 Trop. plant pathol. (2018) 43:21–35

Fig. 4 Phylogenetic relationships of Corynespora isolates based on the consensus tree from a Bayesian analysis of ITS sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted with Corynespora citricola.The isolate obtained from Commelina benghalensis is in bold

Material examined: Brazil, state of Minas Gerais, Viçosa, and phylogenetic differences. Neopyricularia commelinicola Cristais, on living leaves of Commelina benghalensis,20th does not cluster within clades corresponding to species of May 2012, R. W. Barreto (holotype VIC 39065, ex-type cul- Pyricularia s. str. ture COAD 2026). The fungus from TS has straight conidia and 2–4septate Note: The family Pyriculariaceae includes several taxa, which conidiophores whereas P. ebbelsii has curved conidia. The are of great importance as plant pathogens, most notably Brazilian TS fungus is morphologically similar to Magnaporthe and Gaeumannomyces. The best known example Pyricularia oryzae var. commelinae, differing only in the is Pyricularia oryzae (sexual morph Magnaporthe oryzae), the number of septae in its conidiophores (2–4ascomparedto etiological agent of the most important disease of rice – rice blast 1–2-septate in P. oryzae var. commelinae) (Thirumalachar (Skamnioti and Gurr 2009; Klaubauf et al. 2014). Since the initial et al. 1956). Besides that, the newly described fungus has examinations of the fungal structures on diseased TS tissue it was smaller conidia (20–30 × 9–11 μminN. obtusa as compared readily recognized that the fungus associated with these particu- to 27–42 × 10–12.5 μminN. commelinicola) and shorter lar leaf spots was a Pyricularia-like fungus. Four fungi, belong- conidiophores (112–337.5 μminN. obtusa as compared to ing to this group, are known to attack members of the 150–530 μminN. commelinicola) than those of Commelinaceae, namely: Pyricularia ebbelsii - recorded on N. commelinicola. Additionally, conidia in N. obtusa tappers Commelina africana from Tanzania (Ellis 1976); P. grisea - on progressively towards an obtuse apical cell instead of mostly C. benghalensis from Zimbabwe (Whiteside 1966), C. agraria tapering more abruptly at the apical cell, which is long and from Brazil (Purchio and Muchovej 1993)andC. erecta - from almost beak-like in N. commelinicola (Klaubauf et al. 2014). Honduras (Halmos 1970); P. oryzae var. commelinae -on Phylogenetically, the Brazilian fungus clustered in C. benghalensis from India (Thirumalachar et al. 1956; Neopyricularia, in a highly supported clade but separated Hashioka 1973)andNeopyricularia commelinicola - recorded from the type species N. commelinicola (Fig. 5). Therefore, as P. commelinicola on C. communis from Korea (Klaubauf et al. it is being proposed here as a novel species. This is the second 2014). Neopyricularia was recently split from Pyricularia by species described in the recently proposed Neopyricularia ge- Klaubauf et al. (2014) based on a combination of morphological nus. It is interesting to note that the two species in this genus Trop. plant pathol. (2018) 43:21–35 31

Fig. 5 Phylogenetic relationships of Neopyricularia obtusa sp. nov. Tree inferred from Bayesian analysis based on the combined sequences of the ITS and LSU regions. Bayesian posterior probabilities are indicated above the nodes. Magnaporthiopsis maydis represents the outgroup taxon. The isolate obtained from Commelina benghalensis is in bold

are pathogens of members of the Commelinaceae, which jus- leaves and stems. Necrotic tissues covered with flat colonies tifies the collection and molecular analysis of additional of pale brown sterile mycelium, 6–9 μm diam, with constric- Pyricularia-like taxa associated with this plant family, since tions at branch points forming right angles. For a complete they may also belong to Neopyricularia. It is possible that description, see Sneh et al. (1991). Neopyricularia obtusa described here is a fungus originating In culture: Fast-growing (9 cm diam in 4 days), flat, with from Asia and that was introduced in Brazil in the past togeth- entire edges; aerial mycelium felty, with diurnal zonation, er with C. benghalensis, but that remained undetected in honey to buff or white; not sporulating. Brazil until now. A likely origin might be India, where a rather Material examined: Brazil, state of Minas Gerais, Viçosa, similar fungus (perhaps the same taxon) - Pyricularia oryzae campus of the Universidade Federal de Viçosa, on living var. commelinae was described on TS. Additional collections leaves of Commelina benghalensis, 21st September 2012, B. of specimens from India are required in order to confirm or E. C. Miranda (VIC 39066, culture COAD 2028). rule out this hypothesis. Although N. obtusa sometimes is able Notes: On microscopic mounts, orthogonal branching, to cause severe damage to TS, our observations suggest that septation pattern and regular constriction of mature mycelium this is limited to periods when there is a combination of un- were readily observed. These are markers for taxa belonging usually high humidity and low temperatures, when the TS to Rhizoctonia (Sneh et al. 1991). This was further confirmed population tends to naturally decline in the field. by the results of the molecular study of the ITS region. Rhizoctonia solani J. G. Khün, Die Krankheiten der (Fig. 6). A BLAST search yielded 98% homology with FX1 Kulturgewächse: 224. 1858. (Figures1c and 2g). (R. solani AG-4), (GenBank HG934415). Rhizoctonia solani GenBank: ITS sequence KY351637 is a polyphagous pathogen for which more than 2500 host Lesions on living leaves and stems start as irregular soft rot, plants have been reported (Farr and Rossman 2016). necrotic areas along the margins that progress inwards and According to García et al. (2006), 14 anastomosis groups along stems as light brown lesions surrounded by narrow yel- (AG) are recognized in R. solani, AG-1 to AG-13 and AG- lowish haloes. The lesions are fast-growing, coalescent, lead- BI. AG-4, is a group known to lack host-specificity and to ing to a “melted appearance” and destruction of colonized cause seed rot, pre- and post-emergence damping off and 32 Trop. plant pathol. (2018) 43:21–35

Fig. 6 Phylogenetic relationships of Rhizoctonia isolates based on the consensus tree from a Bayesian analysis of ITS sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted with Ceratobasidium stevensii. The isolate obtained from Commelina benghalensis is in bold

occasionally root rot in many crops of economic importance effectiveness in reducing the host population) are to be found in Brazil (Cordeiro and Kimati 1997) and worldwide, as well amongst the natural enemies that remained restricted to the as attacking non-cultivated plants, including weeds (Silva- Indian subcontinent. Barreto et al. 2010). This is the first time R. solani is reported After nearly thirty years of intermittent surveys of fungi on as a pathogen of TS in Brazil and worldwide. Clearly, this TS in Brazil, we conclude that the mycobiota of fungus is inappropriate to be considered for weed biocontrol. C. benghalensis in Brazil is depauperate. Only five fungal pathogens were found and these represented an assemblage dominated by host non-specific pathogens (C. cf sigesbeckiae, R. solani, A. rolfsii and C. cassiicola). Perhaps the only ex- Discussion ception is N. obtusa, but its level of host-specificity still needs to be tested. Besides limited in its diversity, the Brazilian A list of possible fungal and oomycete pathogens, already mycobiota associated with diseased TS does not include any recorded on C. benghalensis, was compiled from the literature biotrophic or semi-biotrophic basidiomycete, such as smut, (Table 3). It includes around 40 taxa. This was expected for smut-like and rust fungi, taxa often regarded as having the such a cosmopolitan weed and is in agreement with what has highest potential for use in classical biological control been found for other pantropical weeds such as Chromolaena (Barreto et al. 2012). Nevertheless, there are records of seven odorata (Barreto and Evans 1994), Mikania micrantha fungal species that belong to such groups and attack (Barreto and Evans 1995b)andLantana camara (Barreto C. benghalensis in the Old World, namely: Kordyana et al. 1995). celebensis, Kordyana indica, Phakopsora tecta, Puccinia Among the taxa included in the list, only three had been commelinae, Uredo ochracea, Bauerago combensis and recorded in the New World. Conversely, there is a significant Bauerago commelinae. diversity of possible TS pathogens (fungi and oomycete gen- The rust fungus Uromyces commelinae represents a puzzle. era known to include plant pathogens) in the Old World, par- It has been recorded on C. benghalensis in India and many ticularly in India, Nepal and Pakistan (the purported center of African countries, but it has never been found on TS in the origin of TS) – 14 species recorded. Evans (1987)considered Americas. Nevertheless, it is of common occurrence in the that several of these fungi show “exceptional promise as bio- Americas on other members of the Commelinaceae. logical control agents”. The best candidates for use in classical Recently, it was found infect T. fluminensis in southern biocontrol (showing host-specificity, host co-evolution and Brazil (Macedo et al. 2016). It is possible that the fungus Trop. plant pathol. (2018) 43:21–35 33

Table 3 List of fungal taxa recorded on (and possibly pathogenic to) Commelina benghalensis worldwide

Fungi Distribuition (country/region) Reference

Zygomycota Choanephora sp. Zambia Riley (1956) Ascomycota Acremonium sp. India Barreto (1991) Alternaria alternata Kenya Caretta et al. (1999) Bipolaris sp. Brazil Mendes et al. (1998)* Bipolaris spicifera Kenya Caretta et al. (1999) Bipolaris victoriae Kenya Caretta et al. (1999) Cercospora apii India Crous and Braun (2003) Cercospora commelinicola Tanzania Ebbels and Allen (1979) Cylindrosporium kilimandscharicum Kenya Mauritius,Tanzania Ebbels and Allen (1979), Nattrass (1961), Orieux and Felix (1968), Cladosporium tenuissimum Kenya Barreto (1991) Cochliobolus hawaiensis India Barreto (1991) Cochliobolus lunatus Kenya Barreto (1991) Cochliobolus verruculosus India Barreto (1991) Colletotrichum dematium India Barreto (1991) Colletotrichum falcatum USA Farr and Rossman (2016) Corynespora cassiicola Samoa Dixon et al. (2009) Curvularia lunata Kenya Caretta et al. (1999) Cylindrosporium kilimandschericum Ethiopia, Kenya, Mauritius, Evans (1987) Nigeria, Sudan, Tanzania, Uganda, Zimbabwe Didymosphaeria sp. Kenya Caretta et al. (1999) Myrothecium roridum India Sinha and Narain (1992) Neopyricularia commelinicola Korea Park and Shin (2009) Nigrospora sphaerica Kenya Caretta et al. (1999) Periconia glyceriicola Kenya Caretta et al. (1999) Phomopsis commelinae Malawi, South Africa, Zimbabwe Evans (1987), Punithalingam (1975) Phomopsis sp. Malawi Corbett (1964) Phyllosticta commelinicola (= Phoma commelinicola) India Mathur (1979) Pseudocochliobolus verruculosus India Evans (1987) Pyricularia grisea Zimbabwe Whiteside (1966) Pyricularia oryzae var. commelinae India, Kenya Evans (1987), Thirumalachar et al. (1956) Septoria commelinae Tanzania Ebbels and Allen (1979) Septoria sp. South Africa Crous et al. (2000) Basidiomycota Athelia rolfsii Tonga Dingley et al. (1981) Bauerago combensis Pakistan Denchev (2003) Bauerago commelinae Pakistan Denchev (2003) Kordyana celebensis Guinea, Kenya, Malawi, Sudan, Ebbels and Allen (1979), Evans (1987), Tanzania, Zambia, Zimbabwe Kranz (1963), Nattrass (1961), Peregrine and Siddiqi (1972), Riley (1956) Kordyana indica India Barreto (1991) Phakopsora tecta Bolivia, Mauritius Evans (1987), Orieux and Felix (1968) Puccinia commelinae Nepal Barreto (1991) Uredo ochracea Philippines Teodoro (1937) Uromyces commelinae Eritrea, India, Indonesia, Castellani and Ciferri (1937), Boedijn (1959), Kenya, Malawi, Papua New Doidge (1950), Ebbels and Allen (1979), Guinea, Southern Africa, Gjaerum (1984), Hosagoudar (1988), Tanzania, Uganda, Zimbabwe Nattrass (1961), Peregrine and Siddiqi (1972), Shaw (1984), Whiteside (1966) Oomycota Phytophthora nicotiana Indonesia Barreto (1991)

*See text for an explanation about this mistaken record 34 Trop. plant pathol. (2018) 43:21–35 causing rust in the Old World on TS is a different species from Barton J (2004) How good are we at predicting the field host-range of the fungus occurring on commelinaceous hosts in the New fungal pathogens used for classical biological control of weeds? Biological Control 31:99–122 World. Another possibility is that strains able to infect Blazquez CH (1967) Corynespora leaf spot of cucumber. Proceedings of C. benghalenis still remain restricted to the Old World. the Florida State Horticultural Society 80:177–182 Another rust fungus was recorded occurring on TS in the Boedijn KB (1959) The Uredinales of Indonesia. Nova Hedwigia 1:463– neotropics (Bolivia) by Evans (1987), although this single 496 Braun U, Sivapalan A (1999) Cercosporoid hyphomycetes from Brunei. record requires confirmation. 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