Electrome alterations in a plant-pathogen system: Toward early diagnosis.

This work aimed to verify the existence of patterns on the electrophysiological systemic responses of tomato plants inoculated with a pathogenic fungus in an environment with controlled light and temperature. Electrical signalling was measured before and after inoculation in the same plants, and data were analysed with time series techniques and approximate multi-scale entropy (ApEn). Machine learning algorithms were utilised in order to classify data before and after infection throughout the five days of experiments. The obtained results have shown that it is possible to distinguish differences in the plant's electrome activity before and after the fungus inoculation. In some cases, we have found scale invariance quantified by the power law decay in the distribution histogram. We also found a higher degree of internal organisation quantified by ApEn. The results of the classification algorithms achieved higher accuracy of infection detection at the initial stage of pathogen recognition by the plant. Besides, this study showed evidence that long-distance electrical signalling is likely involved in the plant-pathogen interaction, since signals were obtained in the stem and the inoculum applied on the plant leaves. This might be useful for the early detection of plant infections.

First Report of Powdery Mildew Caused by Erysiphe platani on Platanus × acerifolia in Rio Grande do Sul, Brazil.

Platanus × acerifolia (Aiton) Willd. (London planetree) is a tree commonly used as an ornamental and in the furniture industry. In the summer of 2013, powdery mildew was observed on shoots of P. × acerifolia plants in the cities of Pelotas and Canela (State of Rio Grande do Sul, Brazil). Voucher specimens (n = 2) were deposited in the Phytopathological Museum Manoel Alves Oliveira at Federal University of Pelotas. Dense white powdery masses of conidia and mycelium were observed on leaves (abaxial and adaxial surfaces), petioles, and young stems. Leaves with high disease severities (≥70%) were deformed with curved edges to the adaxial side, and they often died. Mycelia were superficial with lobed appressoria. Conidiophores were straight, sometimes curved at the base, unbranched, cylindrical, 98 to 236 µm long (137.3 ± 41.2 µm) and composed of a cylindrical foot cell 49 to 102 µm long (66.9 ± 19.5 µm) and 4.4 to 6.4 µm wide (5.3 ± 0.8 µm) followed by two to four cells. Conidia were produced singly or in short chains (two to three), without distinct fibrosin bodies, ellipsoid to ovoid and measuring 24 to 37 µm long (29.5 ± 3.2 µm) and 12 to 19 µm wide (15.2 ± 1.4 µm), often with a wrinkled appearance. Primary conidia had truncate bases and rounded apex while both base and apex were truncated in secondary conidia. Germ tubes were produced apically (pseudoidium type). Chasmothecia were not observed. Genomic DNA was used to amplify the internal transcribed spacer (ITS) region using the ITS1 and ITS4 primers. The resulting sequence (602 bp) was deposited (Accession No. KF499270) in GenBank. BLASTn searches revealed similarity of 100 and 99% with Erysiphe platani from P. orientalis L. (Accession No. JQ365943.1) and P. occidentalis L. (Accession No. JX997805.1), respectively. Phylogenetic analysis placed our sequence in a clade (99% bootstrap support) which included only other E. plantani sequences. In short, morphological and molecular approaches allowed us to identify the infecting fungus as E. platani. For Koch's postulates, 10 detached leaves were inoculated (10 to 15 conidia cm2) on their adaxial surface using an eyelash brush. Non-inoculated leaves served as control. All leaves were kept inside trays with petiole immersed in humidified cotton and maintained at 25 ± 1°C. Symptoms identical to those of the original leaves were observed 6 to 8 days after inoculation, whereas the control leaves remained symptomless. Although E. platani has been previously reported on P. × acerifolia in the city of Poços de Calda, state of Minas Gerais, Brazil (1) and on P. occidentalis in Korea (2), to our knowledge, this is the first record of E. platani on P. × acerifolia in Rio Grande do Sul, Brazil. References: (1) E. M. Inokuti et al. New Dis. Rep. 15:38, 2007. (2) Y. J. La and H. D. Shin. Plant Dis. 97:843, 2013.

Gray Mold Caused by Botryotinia fuckeliana on Edible Pods of Pea in Brazil.

Gray mold on edible pods of snow pea (Pisum sativum Lam. [Fabaceae]) was observed in greenhouse-cultivated pea (cvs. Luana Gigante and Gigante Flor Roxa) in the city of Pelotas (Rio Grande do Sul, Brazil) in September and October 2012. The incidence of diseased pods was high (∼25% of immature pods) after up to 3 cloudy and rainy days that hindered the ventilation inside the greenhouse resulting in high relative humidity. Infection occurred first on senescing petals adhered to the forming pods, leading to pod abortion or rotting that began at the contact site with the infected petal. The first symptoms on pods included water soaked tissue that quickly turned light brown and progressed to necrosis. Conidia and conidiophores produced on profuse gray mycelium could be easily seen on infected tissue 2 to 3 days after the appearance of symptoms. Conidiophores were smooth-walled, 400 µm to over 1.5 mm long, hyaline to pale brown, and branched in their upper part; each branch ended with a hemispherical or spherical swelling, 5 to 9 µm in diameter with minute sterigmata. Macroconidia were globose, ellipsoidal, smooth, hyaline to pale brown, usually with protuberant hila, 7 to 15 × 5 to 9 µm. Microconidia were not observed. On potato dextrose agar (PDA), colonies were fast-growing, white, low, covering entire 10 cm petri plates in 4 to 5 days when they turned gray to brownish-gray. Conidiophores and conidia were often formed in sectors. Shield-like, elliptical, lenticular to irregular, black, 1.5 to 6.0 × 1.0 to 4.0 mm sclerotia developed in 10-day-old colonies incubated at room temperature. Genomic DNA was extracted from conidia, conidiophores, and mycelium and used to amplify both the internal transcribed spacer (ITS) (ITS1-5.8s-ITS2) region and the ß-tubulin gene using the ITS1/4 and Bt2a/b primers, respectively (1,4). The ITS (541 bp) and ß-tubulin (467 bp) sequences were deposited in GenBank under accessions KC683713 and KC683712, respectively. BLASTn searches revealed similarity of 100% (EF207415) and 99% (FQ790278) with Botryotinia fuckeliana (De Bary) Whetzel for the ITS and ß- tubulin sequences, respectively. Based on morphological characteristics and sequence analysis, the pathogen causing pod rot of peas was identified as B. fuckeliana. To fulfill Koch's postulates, 10 unwounded pods of P. sativum 'Luana Gigante' were inoculated by depositing PDA plugs (5 mm) colonized with fungal mycelium on their surface. Non-inoculated and mock-inoculated pods with sterile PDA plugs served as control. Inoculated and control pods were incubated inside a clear plastic box (11 × 11 × 3.5 cm) and over moistened filter paper under 12-h photoperiod at 25 ± 1°C. A surrounding water-soaked halo was visible only on pods inoculated with the fungus 48 h after inoculation (hai). Intense sporulation and necrosis were visible 96 hai. Botrytis spp. was previously detected, through standard blotter test, on seeds of P. sativum in Brazil, but without pathogenicity test nor its transmission through seeds (2,3). To our knowledge, this is the first report of B. fuckeliana causing epidemics on pea pods in Brazil. The high incidence of the disease in a protected environment has the potential to cause significant economic impact due to its damage to the pods, rendering them unmarketable. References: (1) N. L. Glass and G. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) M. A. S. Mendes et al. Fungos em Plantas no Brasil. Embrapa-Cenargen, Brasília, 1998. (3) W. M. Nascimento and S. M. Cícero. Rev. Bras. Sementes 13:5, 1991. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

First Report of Tomato yellow spot virus Infecting Leonurus sibiricus in Brazil.

In Brazil, serious epidemics of begomovirus diseases have been successively reported since the mid-90s, among them those caused by Tomato yellow spot virus (ToYSV) (1). In July 2009 and October 2010, high incidences (40 to 60%) of plants of the weed Leonurus sibiricus (Lamiaceae) exhibiting symptoms of yellow leaf mosaic were found near soybean (Glycine max) crops within the municipalities of Marechal Cândido Rondon and Tapejara, in the states of Paraná and Rio Grande do Sul. Leaves from 21 symptomatic and seven asymptomatic L. sibiricus plants were collected from both localities and tested for the presence of begomovirus. Total DNA was extracted from each sample using Dneasy Plant Mini Kit (Qiagen) and submitted to PCR using begomovirus universal oligonucleotides PAL1v1978/PAR1c496 (3). One fragment of approximately 1,300 bp comprising the 5'-region of the replication-associated protein (Rep) gene, the entire intergenic region (IR), and the 5'-region of the coat protein (CP) gene was amplified from all symptomatic, but not from asymptomatic samples. Amplified fragments corresponding to all isolates were directly sequenced and nucleotide sequence comparisons indicated 98 to 99% nucleotide identity among themselves, and 93 to 94% identity with the corresponding nucleotide sequences for the DNA-A of the begomovirus ToYSV (GenBank Accession No. DQ336350). To confirm these results, the full genome of ToYSV Mc-7 isolated from Marechal Cândido Rondon was cloned and completely sequenced by primer walking (Macrogen Seoul, Korea). The DNA-A of ToYSV Mc-7 (JX513952) was 2,592 nt long and shared 92 and 91% identity with isolates of ToYSV from Argentina (FJ538207) and Brazil (DQ336350), respectively. The DNA-B of ToYSV Mc-7 (JX513952) was 2,568 nt long and shared 91% identity with DNA-B of a Brazilian isolate of ToYSV (DQ336351). The ToYSV Mc-7 isolate is a new strain named Tomato yellow spot virus (Brazil:Marechal Candido Rondon 7:Leonurus:2009) [ToYSV-(BR:MCR7:Le:09)]. To demonstrate pathogenicity, virus-free adults of Bemisia tabaci biotype B were confined on symptomatic L. sibiricus plants for a 48-h acquisition period. The whiteflies were then transferred to healthy L. sibiricus, bean (Phaseolus vulgaris), soybean, and tomato (Solanum lycopersicum) plants. L. sibiricus plants showed the original symptoms on the leaves (five symptomatic plants, seven inoculated plants), whereas bean (3/7), soybean (4/10), and tomato plants (5/10) exhibited mild yellow leaf mosaic. The infection in these symptomatic plants was confirmed by PCR with oligonucleotides PAL1v1978/PAR1c496 (3) and subsequent direct nucleotide sequencing of the 5'-region of the CP gene, which confirmed the identity of the transmitted virus as ToYSV. ToYSV was first reported infecting tomato plants in Minas Gerais state, Brazil (1). Recently, ToYSV was found infecting bean and soybean plants in northwestern Argentina (2). Because L. sibiricus is a weed widely distributed throughout Brazil, and the ToYSV vector B. tabaci is also common, this weed may become a potential source of inoculun of ToYSV to bean, soybean, and tomato crops. To our knowledge, this is the first report of L. sibiricus as a natural host of ToYSV. References: (1) R. F. Calegario et al. Pesq. Agropec. Bras. 42:1335, 2007. (2) P. E. Rodríguez-Pardina et al. Ann. Appl. Biol. 158:69, 2011. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993.

First Report of Powdery Mildew Caused by Golovinomyces sp. on Plantago australis in Brazil.

The plantain Plantago australis Lam. (Plantaginaceae) is a herbaceous species native to southern Brazil that is known for the analgesic, antibiotic, and anti-inflammatory properties of its leaf extracts (2). Powdery mildew was observed on wild P. australis plants in the cities of Tapejara, Jari, and Santa Maria (State of Rio Grande do Sul, Brazil) during the summer of 2011. Affected plants were more often observed in shaded areas. Signs included sparse to abundant white powdery masses of conidia and mycelium on pseudo-petioles and leaves, mostly on the adaxial surface. Severely affected plants (≥80% of foliar area affected) had small chlorotic leaves and reduced size compared to healthy ones. Mycelia were superficial and presented nipple-shaped appressoria. Conidiophores were often curved at the base, unbranched, cylindrical, 81 to 125 µm long (average 97.3 ± 14.9 µm) and composed of a cylindrical foot cell 52 to 73 µm long (average 65.4 ± 7.5 µm) and 9 to 14 µm wide (average 11.6 ± 1.5 µm) followed by one to two shorter cells 17 to 29 µm long (average 23.4 ± 3.6 µm). Conidia were produced in chains of up to eight cells, did not contain fibrosin bodies, ranged from ellipsoid-ovoid to subcylindrical, and measured 24 to 35 µm long (average 30.5 ± 3.7 µm) and 12 to 19 µm wide (average 15.8 ± 1.7 µm). Germ tubes were produced apically (reticuloidium type). Chasmothecia were not observed on sampled leaves. Genomic DNA was extracted from conidia, conidiophores, and mycelium and used to amplify the internal transcribed spacer (ITS) (ITS1-5.8s-ITS2) region using the ITS1 and ITS4 primers. The resulting sequence (558 bp) was deposited under accession number JX312220 in GenBank. Searches with the BLASTn algorithm revealed similarity of 100% with Golovinomyces orontii (Castagne) V.P. Heluta 1988 from Veronica arvensis L. (AB077652.1) (3), 99% with G. orontii from Galium spurium L. and Galium aparine L. (AB430818.1 and AB430813.1) (2) and 99% with G. sordidus (L. Junell) V.P. Heluta 1988 from P. lanceolata L. (AB077665.1) (3). Based on morphological characteristics and sequence analysis of the ITS region, the fungus was identified as belonging to Golovinomyces sp. To fulfill Koch's postulates, five cultivated plants of P. australis with four to five expanded leaves were inoculated by dusting conidia (10 to 15 conidia cm-2) on their leaves. Inoculated and non-inoculated control plants were kept in a greenhouse at 27 ± 5°C and relative humidity of 80 ± 15%. Powdery mildew symptoms identical to those of wild plants were observed 8 to 10 days after in inoculated plants. Although G. sordidus was previously reported on P. australis subsp. hirtella in Argentina and on several species of Plantago in others world regions (1), to our knowledge, Golovinomyces sp. has not been previously reported as a pathogen of P. australis in Brazil. Although the economic impact of the disease is limited, the reduction in plant size and leaves affects biomass production used in the extraction of pharmaceutical compounds. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series 11, 2012. (2) G. C. Sousa et al. J. Ethnopharmacol. 90:135, 2004. (3) S. Takamatsu et al. Mycol. Res. 113:117, 2009.

First Report of Powdery Mildew on Flamboyant Tree Caused by Erysiphe quercicola in Brazil.

Flamboyant (Delonix regia) is an ornamental tree that is native to Madagascar and frequently used in gardens and parks worldwide. Powdery mildew was observed on flamboyant plants in the cities of Piracicaba and São Carlos (State of São Paulo, Brazil) during the springs of 2010 and 2011. All sampled plants (~15 plants) were affected by the disease. Affected plants had abundant, white powdery masses of conidia and mycelium on floral buds that is typical of powdery mildew, but these structures were not observed on leaves and petioles. Diseased buds were observed at all developmental stages. The fungus was identified as Erysiphe quercicola on the basis of scanning electron microscopy, light microscopy, and sequence analysis of the internal transcribed spacer (ITS) region. Conidia were produced in short chains of four to five spores on erect conidiophores. Conidiophores were unbranched, cylindrical, 50 to 80 µm long (mean 68.8 ± 10.8 µm), composed of a cylindrical foot cell 25 to 40 µm long (mean 32.2 ± 4.9 µm), and one to two shorter cells. Conidia were ellipsoid-ovoid to subcylindrical, 22 to 37 µm long (mean 30.9 ± 4.4 µm), and 10 to 18 µm wide (mean 15.1 ± 2.8 µm). Germ tubes were produced apically and ended in a lobed appressorium. Colonizing hyphae also had a well-developed lobed appressorium. Chasmothecia were not observed on buds. DNA was extracted from conidia, conidiophores, and mycelium and used to amplify the ITS (ITS1-5.8s-ITS2) region using the ITS1 and ITS4 primers (2) and its sequence (612 nt) was deposited under Accession No. JQ034229 in the GenBank. Searches with the BLASTn algorithm revealed 100% similarity with E. quercicola from oak (Accession Nos. AB292693.1, AB292691.1, and AB292690.1) (1). To fulfill Koch's postulates, 10 detached young floral buds, 0.4 to 0.8 cm in diameter, were inoculated with five to eight conidia collected on floral buds using an eyelash brush. Inoculated buds were placed on moistened filter paper in petri dishes. The negative control consisted of noninoculated young floral buds. Inoculated and noninoculated buds were incubated in a growth chamber at 25°C and a 12-h photoperiod. Powdery mildew structures were observed 6 to 8 days after inoculation. To our knowledge, E. quercicola has not been reported previously as a pathogen of flamboyant tree since there is no record in the Erysipahales database ( http://erysiphales.wsu.edu/ ). Although the economic impact of the disease is limited, its incidence might induce the abortion of floral buds and accelerate the senescence of flowers, thus reducing the aesthetic value of the trees. References: (1) S. Takamatsu et al. Mycol Res. 111:809, 2007. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.