A rapid rice blast detection and identification method based on crop disease spores' diffraction fingerprint texture.

BACKGROUND: Rice blast fungus is a worldwide disease, and it is one of the most serious rice diseases in the north and south rice fields in China. The initial symptoms of rice blast are not obvious, and the speed of transmission is fast. Manual identification is time-consuming and laborious. At present, it is a great challenge to realize rapid and accurate early identification of rice blast. RESULTS: In this paper, an identification method based on crop disease spores' diffraction fingerprint texture for rice blast was studied; this method utilizes the light field and texture features of diffraction images. To verify the reliability of the model that we proposed, we selected two methods of manual identification and machine recognition to compare and detect rice blast spores. The experimental results show that the identification of light diffraction characteristics is not only higher than the traditional manual recognition by microscope (increased by more than 0.3%), but also faster after neural network training (increased by more than 90%). The diffraction recognition method used in this study, based on crop disease spores' diffraction fingerprint texture, can be completed in a few seconds, and its test accuracy is 97.18%. CONCLUSION: The proposed method, a rapid rice blast detection and identification method based on crop disease spores' diffraction fingerprint texture, has certain advantages compared with the existing manual identification by microscope. This method can be applied to the recognition of rice blast in agricultural research. © 2020 Society of Chemical Industry.

Morphological and molecular characterization of Ceratomyxa batam n. sp. (Myxozoa: Ceratomyxidae) infecting the gallbladder of the cultured Trachinotus ovatus (Perciformes: Carangidae) in Batam Island, Indonesia.

A new coelozoic myxozoan species, Ceratomyxa batam n. sp., was identified in cultured carangid fish, Trachinotus ovatus (Perciformes: Carangidae), in waters off Batam Island of Indonesia. The bi- and trivalved spores were observed in the gallbladder of T. ovatus. Mature bivalved spores of C. batam n. sp. were transversely elongated and narrowly crescent in shape, 3.8 ± 0.36 (2.7-4.6) µm long and 19.2 ± 1.75 (16.2-22.0) µm thick. Two sub-spherical polar capsules were 2.3 ± 0.18 (2.0-2.8) µm long and 2.6 ± 0.16 (2.3-2.9) µm wide. Prevalence was 72.2% in 72 examined T. ovatus according to evaluations dating from November 2016. The maximum likelihood phylogenetic tree based on small subunit rDNA sequence showed similarity with Ceratomyxa robertsthomsoni and Ceratomyxa thalassomae found in Australia. This is the first report of Ceratomyxa species identified in a seawater fish at Batam Island, Indonesia.

New host records of three Kudoa spp. (K. yasunagai, K. thalassomi, and K. igami) with notable variation in the number of shell valves and polar capsules in spores.

To date, 26 Kudoa spp. (Myxozoa: Myxosporea: Multivalvulida) have been recorded in edible marine fishes in Japan. In the future, it is likely that even more marine fish multivalvulid myxosporeans will be characterized morphologically and genetically, which will aid the precise understanding of their biodiversity and biology. We examined 60 individuals of six fish species collected from the Philippine Sea off Kochi or from the border between the Philippine Sea and East China Sea around Miyako Island, Okinawa, i.e., the southern part of Japan. Newly collected parasite species included Kudoa yasunagai from the brain of Japanese meagre (Argyrosomus japonicus) and Japanese parrotfish (Calotomus japonicus), Kudoa miyakoensis n. sp. and Kudoa thalassomi from the brain and trunk muscle, respectively, of bluespine unicornfish (Naso unicornis), and Kudoa igami from the trunk muscle of Carolines parrotfish (Calotomus carolinus), African coris (Coris gaimard), and Pastel ringwrasse (Hologymnosus doliatus). With the exception of Japanese parrotfish for K. yasunagai, all these fish are new host records for each kudoid species. Notable variation in the number of shell valves (SV) and polar capsules (PC) was observed for all four kudoid species. In particular, spores with seven or eight SV/PC were prominent in K. igami isolates, despite the original Japanese parrotfish-derived description characterizing it as having spores with six, or less commonly five, SV/PC. However, molecular genetic characterization based on the ribosomal RNA gene (rDNA) and mitochondrial DNA (cytochrome c oxidase subunit 1 and ribosomal RNA small and large subunits) found no significant differences in the nucleotide sequences of isolates with different phenotypical features as far as examined in the present study. A newly erected species, K. miyakoensis n. sp., was determined to be phylogenetically closest to brain-parasitizing species, such as K. chaetodoni, K. lemniscati, and K. yasunagai based on rDNA nucleotide sequences, but differed from them morphologically.

Ortholinea concentrica n. sp. (Cnidaria: Myxozoa) from the Patagonian seabass Acanthistius patachonicus (Jenyns, 1840) (Perciformes: Serranidae) off Patagonia, Argentina.

The Patagonian seabass Acanthistius patachonicus (Jenyns, 1840) (Serranidae) is a marine fish valued for commercial and sport fisheries from Argentina. We report a new myxosporean (Cnidaria: Myxozoa) infecting the urinary system of the Patagonian seabass from San Antonio Bay, San Matías Gulf, on the Atlantic Ocean. The mature myxospores were subspherical, 8.2-11.0 µm × 7.9-11.0 µm and 7.7-9.0 µm in thickness; two subspherical polar capsules, 2.4-3.8 µm × 2.3-3.6 µm, with 3 to 4 turns of the polar tubule; openings on different valves in almost opposite directions. Ornamented shell valves exhibited 17-20 concentrically organized surface ridges. SSU rDNA phylogenetics analyses placed the new species in the freshwater urinary tract clade, clustering in a clade formed by Myxobilatus gasterostei (Parisi, 1912), Acauda hoffmani Whipps, 2011, and other Ortholinea spp. Based on spore morphology, site of infection, and molecular data, we described this myxozoan as Ortholinea concentrica n. sp.

Morphological plasticity in Myxobolus Bütschli, 1882: a taxonomic dilemma case and renaming of a parasite species of the common carp.

BACKGROUND: Myxozoans are a group of cnidarian parasites, the present taxonomy of which favors a more comprehensive characterization strategy combining spore morphology, biological traits (host/organ specificity, tissue tropism), and DNA data over the classical morphology-based taxonomy. However, a systematist might again run into a taxonomic dilemma if more than two of the following exceptional cases were encountered at the same time: extensive intraspecific polymorphism, interspecific morphological similarity, identical interspecific biological traits and blurred small-subunit (SSU) rDNA-based species boundaries. In the present study, spores of a species of Myxobolus Bütschli, 1882 with two morphotypes (wide type and narrow type) were collected from the gills of common carp Cyprinus carpio Linnaeus. Confusingly, the wide type was found to be identical to Myxobolus paratoyamai Kato, Kasai, Tomochi, Li & Sato, 2017 in spore morphology and SSU rDNA sequence, which confidently suggested their conspecificity; while the narrow type, was highly similar to Myxobolus toyamai Kudo, 1917 based on spore morphology and SSU rDNA sequence and thus could not be easily classified. This discordance between wide type and narrow type has caused a taxonomic dilemma. To address this problem, a hypothesis about the conspecificity of the narrow type and M. toyamai was addressed. RESULTS: It was found that if the narrow type is conspecific with M. toyamai, it would be paradoxical for the SSU rDNA sequence of the narrow type to be more similar to M. paratoyamai (99.3%), Myxobolus acinosus Nie & Li, 1973 (98.6%) and Myxobolus longisporus Nie & Li, 1992 (98.7%) than to M. toyamai (97.6%). According to the results of the above what-if analysis, the narrow type and M. toyamai were considered to be different species. All in all, the present dual-morphotype species is estimated to be conspecific with M. paratoyamai Kato, Kasai, Tomochi, Li & Sato, 2017. Considering that this species name was preoccupied by Myxobolus paratoyamai Nie & Li, 1992, the replacement name Myxobolus pseudoacinosus nom. nov. is proposed. CONCLUSIONS: This work addresses the taxonomic dilemma in polymorphic myxozoans and demonstrates that M. pseudoacinosus is a distinct species with two morphotypes. The present study may serve as a baseline for future studies that encounter similar classification complexities.

Morphological and molecular analyses of Bipteria lusitanica n. sp. in wild white seabream, Diplodus sargus (Linnaeus, 1758) in Portugal.

The present study records the presence of Bipteria lusitanica n. sp. in wild white seabream, Diplodus sargus (Linnaeus, 1758) off the Portuguese coast. Myxosporean parasites were found in the interstitial tissue of kidney and free in urinary bladder of D. sargus with a 33.3% of prevalence of infection. Myxospores were triangular or inversely pyramidal in shape and anteriorly wider in frontal view, measuring 11.2 ± 1.0 µm in length, 12.6 ± 0.9 µm in width, and 11.6 ± 0.4 µm in thickness. The sinuous sutural line was parallel to the axis that connects the center of the two polar capsules. Spore valves were smooth without ridges. Wing-like appendages extended from the posterior part of each valve in sutural view, measuring 3.9 ± 1.1 µm in length. Spherical polar capsules measured 4.4 ± 0.2 µm in diameter and the polar filaments were helical arranged in 5-6 turns. Molecular data showed that this parasite clusters within species of the Sinuolineidae Shulman, 1959 family, and they all infect the urinary bladder. Using molecular and morphological characterization, we were able to identify this parasite as a novel species of the genus Bipteria Kovaleva, Zubtchenko, and Krasin, 1983.

Ceratomyxa azevedoi n. sp. (Myxozoa: Myxosporea) parasitizing the gallbladder of Lutjanus ehrenbergii in the Arabian Gulf.

A novel myxosporean species, Ceratomyxa azevedoi sp. n. is described from the gallbladder of the blackspot snapper, Lutjanus ehrenbergii (Peters), captured from the Arabian Gulf off Saudi Arabia. A total of 45 (26.8%) out of 168 fish specimens were found to be infected with Ceratomyxa azevedoi sp. n., the highest prevalence being observed in winter (42.9%, 18/42) and the lowest in autumn (11.9%, 5/42). Mature spores appeared as crescent to slightly elliptical-shaped, measuring 5-7 (6) µm in length and 12 (10-14) µm in thickness, with spherical polar capsules containing three polar filament coils. The morphometric and morphological comparison with similar species revealed the taxonomic novelty of this form, suggesting that it should be considered as new species. The phylogenetic analysis of C. azevedoi sp. n., based on partial SSU rDNA sequences, revealed close genetic relatedness to C. buri with 91.3% homogeneity and to C. hamour, with 90.1% homogeneity.

Revision of series Gravesiana (Adiantum L.) based on morphological characteristics, spores and phylogenetic analyses.

Since the adoption of some ambiguous and quantitative characters in Flora Republicae Popularis Sinicae 3(1), species identifications of the series Gravesiana have been in disarray, requiring clarification. Two hundred and fifty-nine individuals from 47 different populations were collected for the estimation of morphological characters and phylogenetic analyses. Spores of 26 populations were observed through scanning electron microscope. Our results were different from those of previous research: (1) six identifiable species, rather than five species observed previously, were confirmed in the series Gravesiana, they are A. gravesii, A. juxtapositum, A. mariesii, A. dentatum, A. longzhouensis and A. obovatum, of which the latter three are newly recognized species. (2) Thirteen characters were measured and estimated through the program Mesquite v. 2.71. The character whether the pinna stalks were 1/3-1/2 times longer than the pinna was used to distinguish A. gravesii and A. lianxianense previously and was found to be unreliable here, whereas such characters as the height of the plant (H), pinna aligned forms (FP), number of pinna (NP), pinna margin (M), number of veins flabellate at base (NV), sori number and shape per pinna (NSS), pinna texture (T), and powder-covered or not on the abaxial surface of the pinna (P) are estimated to be stable and reliable characters useful for identification. Descriptions of new species and their retrieve keys are also listed. (3) Surface ornamentations and spore sizes are helpful for us to distinguish species in series Gravesiana.

Occurrence of two novel actinospore types (Cnidaria: Myxosporea) in Brazilian fish farms, and the creation of a novel actinospore collective group, Seisactinomyxon.

The involvement of oligochaetes in the life cycles of fresh water myxozoan parasites in Brazil was investigated. Of 333 oligochaetes collected in a fish farm in the State of São Paulo, three (0.9%) released Aurantiactinomyxon type spores. From 86 worms collected in a fish farm in Mato Grosso do Sul State, 1 (0.9%) released actinospores with a novel morphology for which we propose the name Seisactinomyxon. Infected oligochaetes were identified by morphology: all belonged to family Naididae, with Pristina americana the host for Aurantiactinomyxon and Slavina evelinae the host of Seisactinomyxon. This is the first report of the involvement these two species of oligochaetes in the life cycle of myxozoans. Small subunit rDNA sequences of the Aurantiactinomyxon (1204 nt) and Seisactinomyxon (1877 nt) did not match any previously sequenced myxozoan. Phylogenetic analyses showed that both actinospore types fell in a clade formed by six Myxobolus spp. that parasitize Characiformes fishes.

Comparison of pollen levels between 2 pollen traps in Salamanca, Spain

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Ultrastructure and ssrRNA sequencing of Myxidium amazonense n. sp. a myxosporean parasite of Corydoras melini from the Rio Negro river, Amazonas state, Brazil.

In a survey of myxozoan parasites of ornamental freshwater fish from the Rio Negro river, it was found that seven of 30 (23.3 %) Corydoras melini specimens examined had plasmodia of a new Myxidium species (Myxidium amazonense n. sp.) in the gallbladder. The fish were caught in the Rio Negro river, in the municipality of Santa Isabel do Rio Negro, in the state of Amazonas, Brazil. The plasmodia had a tubular shape, which was organized as a spiral spring with several turns in the gallbladder. The development of the myxospores was asynchronic, with disporic pansporoblasts. Mature myxospores were elongated, with 17.0 ± 0.9 (16.1-17.9) µm in length and 3.7 ± 0.7 (3.0-4.4) µm in width, and lightly arcuate from the valval view, with their bodies tapering slowly until ending in rounded extremities. The valval surface had nine to ten grooves in each valve. The polar capsules, one at either end of the spore, had a length of 5.4 ± 0.5 (4.9-5.9) µm and a width of 3.4 ± 0.6 (2.8-4.0) µm. Ultrastructural analysis showed that the wall of the plasmodia had numerous microvilli-like structures, pinocytotic canals, and cytoplasmic bridges connecting the pansporoblasts to each other and to the ectoplasm zone. Phylogenetic analysis, based on a small subunit ribosomal RNA (ssrRNA), identified the new species as a sister species of Myxidiumceccarelli, the unique South American Myxidium species whose ssrRNA sequence is available in the NCBI database. This study is the first description of Myxidium species in ornamental freshwater fish from Amazon.

Small subunit ribosomal RNA sequence links the myxospore stage of Henneguya mississippiensis n. sp. from channel catfish Ictalurus punctatus to an actinospore released by the benthic oligochaete Dero digitata.

There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 µm (range = 40.7-61.6 µm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 µm (14.4-19.3 µm) in length and 5.0 ± 0.3 µm (4.5-5.5 µm) in width. The two polar capsules were 6.2 ± 0.4 µm (5.8-7.0 µm) long and 5.0 ± 0.3 µm (4.5-5.5 µm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 µm (22.9-40.6 µm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4% similarity (100% coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.

A new isolate of Amoeboaphelidium protococcarum, and Amoeboaphelidium occidentale, a new species in phylum Aphelida (Opisthosporidia).

Microalgae used in the production of biofuels represents an alternative to fossil fuels. One problem in the production of algae for biofuels is attacks by algal parasitoids that can cause population crashes when algae are cultivated in outdoor ponds (Greenwell et al. 2010). Integrated solutions are being sought to mitigate this problem, and an initial step is pest identification. We isolated an algal parasitoid from an open pond of Scenedesmus dimorphus used for biofuel production in New Mexico and examined its morphology, ultrastructure and molecular phylogeny. A phylogenetic analysis placed this organism in Aphelida as conspecific with Amoeboaphelidium protococcarum sensu Karpov et al. 2013. As a result we re-evaluated the taxonomy of Amoeboaphelidium protococcarum sensu Letcher et al. 2013 and here designate it as a new species, Amoeboaphelidium occidentale.

Molecular and morphometric characteristics of Ceratomyxa hamour n. sp. (Myxosporea: Bivalvulida) infecting the gallbladder of the orange-spotted grouper Epinephelus coioides from the Arabian Gulf, Saudi Arabia.

Ceratomyxa hamour n. sp. was found to infect the gallbladder of the orange-spotted grouper, Epinephelus coioides located off the Saudi Arabian coast of the Arabian Gulf. The infection was reported as a free-floating spore in the bile, and pseudoplasmodia were not observed. Mature spores were crescent-shaped and measured on average 7 µm in length and 16 µm in thickness. The polar capsule, meanwhile, had length to width measurements of 4 µm and 3 µm on average. A periodical survey was conducted throughout a sampling period between December 2012 and December 2013, with the results showing that the parasite was present throughout the year with a mean prevalence of 32.6%. The objective of this study was to characterize this new species based on its morphological and molecular differences from previously described species. Molecular analysis based on the partial sequence of the SSU rDNA gene, showed the highest similarity (97.8%) to Ceratomyxa buri, reported in the cultured yellow tail Seriola quinqueradiata in Japan. Indeed, C. buri and the new species described here formed an individual cluster with a high degree of bootstrap support. This is the first reported species of genus Ceratomyxa from the Arabian Gulf fishes off Saudi Arabia.

[Morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius: a contribution to the systematics of Neotropical Lycopodiaceae]. / Morfología y anatomía de ejes caulinares, licofilos y esporangios de Phlegmariurus phylicifolius: un aporte a la sistemática de las Lycopodiaceae neotropicales.

Phlegmariurus is the only genus of Lycopodiaceae with the species grouped in 22 informal groups. Species level relationships within Phlegmariurus are poorly understood and their circumscriptions require a thorough molecular and morphological review. A detailed study of morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius was carried out in order to contribute to the elucidation of species circumscription in the informal group Phlegmariurus phlegmaria. Small pieces of caulinar axes bearing trophophylls, sporophylls and sporangia were fixed, dehydrated, Histowax (paraffin) embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made and stained with the same technique. P. phylicifolius includes slender, pendulous plants up to 40cm long. Shoots heterophyllous, in the basal divisions ca. 10-20(-25) mm in diameter including the trophophylls, then abruptly constricted to (1-) 1.5-2(-2.5) mm in diameter including the imbricate, reduced sporophylls. Trophophylls are borne in alternating whorls of three, or decussate, subdecussate, or alternate, widely spaced in alternate leaved caulinar axes portions, perpendicular to the caulinar axes to falcately ascending, lanceolate to linear-lanceolate, with flat to slightly revolute margins. Each lycophyll is supplied by a single central vascular bundle, connected to a protoxylem pole in the stele. At the site of leaf-trace departure, no leaf (lycophyll) gap is present. Caulinar axes excluding leaves 0.7-1.2 mm thick at the base, upward tapering to ca. 0.5 mm. Caulinar axes present unistratified epidermis and endodermis, the cortex is characterized by the presence of a trabecular structure of lisigenous origin formed in the parenchimatous tissue next to the endodermis. The vascular tissue occupies the central part of the caulinar axes, forming a plectostele ofsubradiate organization, with five poles ofprotoxylem. The epidermal cells present sinuous anticlinal walls; invaginations in the inner side of external periclinal wall of the epidermal cells could be probably adaptive morphological feature of a water deficient environment. Leaves of constricted terminal divi- sions are decussate, or subdecussate, continuously or discontinuously sporangiate, appressed, abaxially rounded to carinate, widely lanceolate to widely ovate or subcordate, acute to mucronate or cuspidate, shorter than the sporangia. Each sporangium originates from a group of epidermal cells, axilar to the sporophylls. The cell walls of epidermal cell of the sporangia are Huperzioideae type. The morphological studies of trophophylls contribute to confirm the differences between P. phylicifolius and P. subulatus.

Morfología y anatomía de ejes caulinares, licofilos y esporangios de Phlegmariurus phylicifolius: un aporte a la sistemática de las Lycopodiaceae neotropicales / Morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius: a contribution to the systematics of Neotropical Lycopodiaceae

Phlegmariurus is the only genus of Lycopodiaceae with the species grouped in 22 informal groups. Species level relationships within Phlegmariurus are poorly understood and their circumscriptions require a thorough molecular and morphological review. A detailed study of morphology and anatomy of caulinar axes, lycophylls and sporangia of Phlegmariurus phylicifolius was carried out in order to contribute to the elucidation of species circumscription in the informal group Phlegmariurus phlegmaria. Small pieces of caulinar axes bearing trophophylls, sporophylls and sporangia were fixed, dehydrated, Histowax (paraffin) embedded, sectioned in a rotatory microtome, and stained using the common Safranin O-Fast Green technique; handmade cross sections were also made and stained with the same technique. P. phylicifolius includes slender, pendulous plants up to 40cm long. Shoots heterophyllous, in the basal divisions ca. 10-20(-25)mm in diameter including the trophophylls, then abruptly constricted to (l-) 1.5-2(-2.5)mm in diameter including the imbricate, reduced sporophylls. Trophophylls are borne in alternating whorls of three, or decussate, subdecussate, or alternate, widely spaced in alternate leaved caulinar axes portions, perpendicular to the caulinar axes to falcately ascending, lanceolate to linear-lanceolate, with flat to slightly revolute margins. Each lycophyll is supplied by a single central vascular bundle, connected to a protoxylem pole in the stele. At the site of leaf-trace departure, no leaf (lycophyll) gap is present. Caulinar axes excluding leaves 0.7-1.2mm thick at the base, upward tapering to ca. 0.5mm. Caulinar axes present unistratified epidermis and endodermis, the cortex is characterized by the presence of a trabecular structure of lisigenous origin formed in the parenchimatous tissue next to the endodermis. The vascular tissue occupies the central part of the caulinar axes, forming a plectostele of subradiate organization, with five poles of protoxylem. The epidermal cells present sinuous anticlinal walls; invaginations in the inner side of external periclinal wall of the epidermal cells could be probably adaptive morphological feature of a water deficient environment. Leaves of constricted terminal divisions are decussate, or subdecussate, continuously or discontinuously sporangiate, appressed, abaxially rounded to carinate, widely lanceolate to widely ovate or subcordate, acute to mucronate or cuspidate, shorter than the sporangia. Each sporangium originates from a group of epidermal cells, axilar to the sporophylls. The cell walls of epidermal cell of the sporangia are Huperzioideae type. The morphological studies of trophophylls contribute to confirm the differences between P. phylicifolius and P. subulatus. Rev. Biol. Trop. 62 (3): 1217-1227. Epub 2014 September 01.

Phlegmariurus es el único género de Lycopodiaceae con las especies reunidas en 22 grupos informales. Las relaciones a nivel de especie dentro de Phlegmariurus están pobremente estudiadas y la circunscripción de las mismas requiere profundos exámenes moleculares y morfológicos. Se ha llevado a cabo un estudio detallado de la morfología y la anatomía de ejes caulinares, licofilos y esporangios de P. phylicifolius, con el fin de contribuir al esclarecimiento en la delimitación de las especies en el grupo Phlegmariurus phlegmaria. Segmentos de ejes caulinares con trofofilos, esporofilos y esporangios fueron fijados, deshidratados, incluidos en Histowax (parafina), cortados con un micrótomo rotatorio y coloreados usando la técnica tradicional Safranina O-Verde Rápido; además se hicieron cortes a mano alzada y se colorearon con la misma técnica. P. phylicifolius incluye plantas colgantes y péndulas de hasta 40cm de longitud. Los ejes son heterofilos, de aproximadamente 10-20(-25)mm de diámetro en las divisiones basales incluyendo los trofofilos, luego abruptamente reducidos a (l-) 1.5-2(-2.5)mm de diámetro incluyendo los esporofilos reducidos e imbricados. Los trofofilos están dispuestos en anillos alternantes de a tres, o decusados, subdecusados o alternos, dispuestos en forma espaciada en los ejes caulinares, perpendiculares al tallo hasta falcadamente ascendentes, lanceolados a lineal-lanceolados, con márgenes lisos o levemente revolutos. Cada licofilo está provisto de un haz vascular simple y central, conectado a un polo de protoxilema de la estela y sin laguna foliar. Los tallos poseen un ancho de 0.7-1.2mm en la base, excluyendo los licofilos, estrechándose hasta cerca de 0.5mm hacia el ápice. Los ejes caulinares presentan una epidermis uniestratificada y endodermis, la corteza se caracteriza por la presencia de una estructura trabecular de origen lisígeno formada en el tejido parenquimático próximo a la endodermis. El tejido vascular ocupa la parte central del eje caulinar, formando una plectostela de organización subradiada, con cinco polos de protoxilema. Las células epidérmicas presentan paredes anticlinales sinuosas; las invaginaciones en la cara interna de la pared periclinal externa podrían ser probablemente un característica morfológica adaptativa a un ambiente con períodos de sequía. Las hojas de las porciones apicales son decusadas o subdecusadas, con esporangio de disposición continua o discontinua, adpresas, abaxialmente redondeadas a carinadas, ampliamente lanceoladas a ovadas o subcordadas, ápice agudo a mucronado o cuspidado, más corto que el esporangio. Cada esporangio se origina de un grupo de células epidérmicas, en la axila de los esporofilos con el eje caulinar. Las paredes celulares de las células epidérmicas del esporangio son de tipo Huperzioideae. El estudio de la morfología de los trofofilos contribuye a confirmar las diferencias entre P. phylicifolius y P. subulatus.

[Ontogeny of the sporangium, spore formation and cytochemistry in Colombian Lycopodials (Lycopodiaceae)]. / Ontogenia de los esporangios, formación y citoquímica de esporas en licopodios (Lycopodiaceae) colombianos.

Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5 microm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5 microm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.

Ontogenia de los esporangios, formación y citoquímica de esporas en licopodios (Lycopodiaceae) colombianos / Ontogeny of the sporangium, spore formation and cytochemistry in Colombian Lycopodials (Lycopodiaceae)

Studies on reproductive aspects of Lycopodiaceae are not very abundant in the scientific literature, and constitute essential information to support taxonomic and systematic relationships among the group. Here we present a detailed study of the ontogeny of sporangia and sporogenesis, and the chemical determination of several compounds generated during spore formation. The analyses were performed in 14 taxa of six genera of the family, Diphasiastrum, Diphasium, Huperzia (a genus which is treated here including Phlegmariurus), Lycopodiella, Lycopodium and Palhinhaea. Specimens were collected in three departments from the Colombian Andes between 1 454-3 677m altitude. Ontogeny was studied in small, 1cm long pieces of strobili and axis, which were fixed in glutaraldehyde or FAA, dehydrated in alcohol, embedded in LR White, sectioned in 0.2-0.5μm and stained with toluidine blue (TBO), a metachromatic dye that allows to detect both sporopollenin and lignin or its precursors, during these processes. For other studies, paraplast plus-embedded sections (3-5μm) were stained with safranin-fast green and alcian blue-hematoxylin. Chemical tests were also conducted in sections of fresh sporangia at different stages of maturity using alcian blue (mucopolysaccharides), Lugol solution (starch), Sudan III (lipids), phloroglucinol (lignin) and orcein (chromosomes). Sections were observed with photonic microscope equipped with differential interference contrast (DIC) and fluorescence microscopy (for spore and sporangium walls unstained). Strobili and sporangia were dehydrated with 2.2 dimethoxypropane, critical point dried and coated with gold for scanning electron microscopy (SEM). Our results indicated that the ontogeny of sporangia and sporogenesis were very similar to the previously observed in Huperzia brevifolia. Cutinisation occurs in early stages of development of sporangium cell walls, but in their final stages walls become lignified. As for the sporoderm development, the exospore is the first layer formed, composed by sporopollenin. The endospore deposits as a thin inner layer composed of cellulose, pectin and carboxylated polysaccharides. The perispore, if present, deposits at last. Mucopolysaccharides were found on the sporocyte coat and its abundance in sporangial cavity persists up to the immature tetrads stage, and then disappears. The lipids were abundant in the sporocytes, tetrads and spores, representing the main source of energy of the latter. In contrast, starch is not detected in the spores, but is abundant in premeiotic sporocytes and immature tetrads, developmental stages of high cellular metabolic activity. Intrinsic fluorescence corroborates the presence of lignin and cutin in the sporangium wall, while the sporopollenin is restricted to the exospore. The transfusion cells and the perispore are not always present. However, the processes of ontogeny and sporogenesis are extremely similar throughout the taxa studied, suggesting that they represent conservative family traits, nonspecific or generic.

Los estudios sobre aspectos reproductivos no son muy abundantes en la literatura científica sobre los taxones de Lycopodiaceae y constituyen información esencial para apoyar la taxonomía y relaciones sistemáticas en el grupo. Por lo tanto, se presenta aquí un análisis detallado de la ontogenia de los esporangios y esporogénesis, así como determinaciones químicas de varios compuestos generados durante la formación de las esporas. Los análisis se llevaron a cabo en 14 taxones de seis géneros de la familia: Diphasiastrum, Diphasium, Huperzia (un género que se trata aquí, incluyendo Phlegmariurus), Lycopodiella, Lycopodium y Palhinhaea. Las muestras fueron recolectadas en tres departamentos de los Andes de Colombia entre 1 454-3 677m de altitud. La ontogenia se estudió en trozos de estróbilos y ejes, de 1cm de largo, que se fijaron en glutaraldehido o FAA, se deshidrataron en alcohol, se incluyeron en LR White, se seccionaron en cortes de 0.2-0.5μm y se colorearon con azul de toluidina (TBO), un colorante metacromático que permite detectar tanto esporopolenina como lignina o sus precursores. Para estudios adicionales, secciones de 3-5μm de material incluido en paraplast plus se colorearon con safranina-verde rápido y azul alciánhematoxilina. Las pruebas químicas se llevaron a cabo en secciones de esporangios sin fijar en diferentes etapas de madurez utilizando azul alcián (mucopolisacáridos), solución de Lugol (almidón), Sudán III (lípidos), fluoroglucinol (lignina) y orceína (cromosomas). Las observaciones se efectuaron con microscopio fotónico equipado con contraste diferencial de interferencia (DIC) y microscopía de fluorescencia (para esporas y pared de los esporangios sin colorear). Para observaciones con microscopía electrónica de barrido (MEB), los estróbilos y esporangios se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Los resultados indican que la ontogenia de los esporangios y esporogénesis es muy similar a la observada previamente en Huperzia brevifolia. En las primeras etapas de desarrollo, las paredes celulares de la epidermis del esporangio se cutinizan y en las finales se lignifican. En el desarrollo del esporodermo, la primera capa que se forma es el exosporio, compuesto por esporopolenina. El endosporio es una capa interna delgada compuesta de celulosa, pectina y polisacáridos carboxilados. El perisporio, si está presente, es la última capa que se deposita. Los mucopolisacáridos se encontraron en la cubierta del esporocito, son abundantes en la cavidad esporangial hasta la etapa de tétradas inmaduras y luego desaparecen. Los lípidos son abundantes en esporocitos, tétradas y esporas, y representan la principal fuente de energía de estas. En contraste, el almidón no se detecta en las esporas pero es abundante en esporocitos premeióticos y tétradas inmaduras, ambos con gran actividad metabólica. La fluorescencia intrínseca corrobora la presencia de lignina y cutina en la pared del esporangio, mientras que la esporopolenina se limita al exosporio. Las células de transfusión y el perisporio no siempre están presentes. Sin embargo, los procesos de la ontogenia y esporogénesis son extremadamente similares en todos los taxones estudiados, lo que sugiere que representan rasgos típicos de familia, no específicos ni genéricos.

Intact cell/intact spore mass spectrometry (IC/ISMS) on polymer-based, nano-coated disposable targets.

Identification and differentiation of microorganisms has and still is a long arduous task, involving culturing of the organism in question on different growth media. This procedure, which is still commonly applied, is an established method, but takes a lot of time, up to several days or even longer. It has thus been a great achievement when other analytical tools like matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were introduced for faster analysis based on the surface protein pattern. Differentiation and identification of human pathogens as well as plant/animal pathogens is of increasing importance in medical care (e.g. infection, sepsis, and antibiotics resistance), biotechnology, food sciences and detection of biological warfare agents. A distinction between microorganisms on the species and strain level was made by comparing peptide/protein profiles to patterns already stored in databases. These profiles and patterns were obtained from the surface of vegetative forms of microorganisms or even their spores by MALDI MS. Thus, an unknown sample can be compared against a database of known pathogens or microorganisms of interest. To benefit from newly available, metal-based disposable microscope-slide format MALDI targets that promise a clean and even surface at a fraction of the cost from full metal targets or MTP (microtiter plate) format targets, IC/ISMS analysis was performed on these and the data evaluated. Various types of bacteria as well as fungal spores were identified unambiguously on this disposable new type of metal nano-coated targets. The method even allowed differentiation between strains of the same species. The results were compared with those gained from using full metal standard targets and found to be equal or even better in several aspects, making the use of disposable MALDI targets a viable option for use in IC/ISMS, especially e.g. for large sample throughput and highly pathogenic species.

Proposed changes to the nomenclature of Ichthyophonus sp. life stages and structures.

Much of the terminology describing Ichthyophonus sp. life stages and structures can be traced to the mistaken classification of this organism as a fungus. This misidentification led early investigators to use mycological terms for the structures they observed; while some terminology is not so easily explained, it appears to have been co-opted from the fields of botany and bacteriology. The purpose of this exercise is to attempt to standardize the terminology associated with Ichthyophonus and to bring it into agreement with terminology currently used to define similar life stages of other protists. The proposed changes are (1) spore/macrospore/mother spore to "schizont," (2) microspore/endospore to "merozoite," and (3) pseudohyphae to "hyphae" or "germ tube."