Use of cereals as basal medium for the formulation of alternative culture media for fungi

Summary The feasibility of developing alternative media to different culture media particularly potato dextrose agar was assessed using local cereal species as the basal media. Three cereal meal extracts – corn, sorghum and millet – were prepared, using them as substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard set up with which the performances of the formulated media were compared. Eight genera of fungi (Aspergillus niger, Fusarium moniliforme, Penicillium sp., Cercospora sp., Curvularia palescens, Botryodiplopodia sp., Rhizopus sp. and Rhodotorula rubra) were isolated and pure cultures of each species aseptically inoculated onto the three different formulated media including PDA and allowed to grow. Their growths were measured at 24, 48, 72, and 96 h after inoculation, using diameter of growth as an index. The set up was repeated thrice for each species on the three formulated media and the control (PDA). Growth of all the fungal species were observed to be about the same or sometimes better in the formulated media relative to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F. moniliformehad an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at 96 h while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 + 1.16 mm at 48 h. Botryodiplopodia sp. grew through the whole diameter of the plate (covering the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 h.     

Decomposition of cellulose strips in relation to climate,litterfall nitrogen,phosphorus and C/N ratio in natural boreal forests

Isolates of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum, Penicillium sp., Rhizoctonia solani, Stemphylium sp., Thielaviopsis basicola, and Verticillium dahliae were cultured on potato–dextrose agar (PDA), barley-sand and alfalfa-sand substrates in petri-dish or in column microcosms. N-mineralization by fungi and fungal-feeding nematodes in combination or fungi alone was assessed. Numbers of Aphelenchus avenae or Aphelenchoides composticola supported by the fungi were measured every 7 days. Times for full colonization of the substrates by fungi ranged from 5 to 15 days. Rhizoctonia solani and B. cinerea on PDA supported the largest A. avenae and A. composticola populations, respectively. Penicillium sp. was a nonhost for A. composticola and A. avenae. Rhizoctonia solani, B. cinerea, V. dahliae, and F. oxysporum supported significantly more nematodes than the other four fungal species. The ranked order of fungi based on the amount of N mineralized in columns free of nematodes was A. alternata (with a rate of 0.052 μg N/g-sand per day), Stemphylium sp., V. dahliae, T. basicola, B. cinerea, F. oxysporum, R. solani, and Penicillium sp. (with a rate of 0.0045 μg N/g-sand perday). The presence of A. avenae resulted in significant increases in mineral N, compared to nematode-free columns colonized by F. oxysporum, R. solani, and T. basicola alone. The presence of A. composticola resulted in significant increases in mineral N, compared to nematode-free columns colonized by A. alternata, B. cinerea, F. oxysporum, and R. solani alone. There was more mineral N incolumns in the presence of A. composticola than A. avenae in most cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation and characterisation of phosphate solubilising microorganisms from the cold desert habitat of <Emphasis Type="Italic">Salix alba Linn.</Emphasis> in trans Himalayan region of Himachal Pradesh

Phosphate solubilising microorganisms (PSM) (bacteria and fungi) associated with Salix alba Linn. from Lahaul and Spiti valleys of Himachal Pradesh were isolated on Pikovskaya (PVK), modified Pikovskaya (MPVK) and National Botanical Research Institute agar (NBRIP) media by spread plating. The viable colony count of P-solubilising bacteria (PSB) and fungi (PSF) was higher in rhizosphere than that of non-rhizosphere. The frequency of PSM was highest on MPVK followed by NBRIP and PVK agar. The maximum proportion of PSM out of total bacterial and fungal count was found in upper Keylong while the least in Rong Tong. The PSB frequently were Gram-positive, endosporeforming, motile rods and belonged to Bacillus sp. The PSF mainly belonged to Penicillium sp., Aspergillus fumigatus, A. niger, A. spp. and non-sporulating sterile. Amongst the isolates with high efficiency for tricalcium phosphate (TCP) solubilisation, seven bacterial and seven fungal isolates dissolved higher amount of P from North Carolina rock phosphate (NCRP) than Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). However, the organisms solubilised higher-P in NBRIP broth than PVK broth. SBC5 (Bacillus sp.) and SBC7 (Bacillus sp.) bacterial isolates exhibited maximun P solubilisation (40 and 33 μg ml⁻¹ respectively) whereas FC28 (Penicillium sp.) isolate (52.3 μg ml⁻¹) amongst fungi while solubilising URP. The amount of P solubilised was positively correlated with the decrease in pH of medium. SBC5 (Bacillus sp.), SBC7 (Bacillus sp.) and SBC4 (Micrococcus) decreased the pH of medium from 6.8 to 6.08 while FC28 (Penicillium sp.) and FC39 (Penicillium sp.) isolates of fungi recorded maximum decrease in pH of medium from 6.8 to 5.96 in NBRIP broth.     

Anaerobic growth ability and alcohol fermentation activity of microscopic fungi

The method proposed in this study was used to isolate fungi grown under anaerobic conditions and to reveal distinctions in their abundance and species composition in different habitats. The ability of micromycetes of different taxa to grow under anaerobic conditions and ensure alcohol fermentation was determined for a representative sample (344 strains belonging to more than 60 species). The group of fungi growing under anaerobic conditions included species with high, moderate, and low fermentation activity. The ability for anaerobic growth and fermentation depended on the taxonomic affiliation of fungi. In some cases, the expression of these characteristics depended on the habitat from which the strain was isolated. The maximum level of ethanol accumulation in culture liquid (1.2–4.7%) was detected for Absidia spinosa, Aspergillus sp. of group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp.     

Root-surface mycoflora of cassava (Manihot esculenta) and post harvest rot of the tubers

The root-surface mycoflora of cassava were isolated from roots washed in serial changes of sterile distilled water and plated out on potato-dextrose agar. A small group of fungi which included Aspergillus niger, Botryodiplodia theobromae, Fusarium solani, Penicillium javanicum, Penicillium sp., and Trichoderma sp. were found to be consistently associated with the root surface. While the isolates, B. theobromae and F. solani were found to be aggressively pathogenic on freshly harvested cassava tubers causing extensive rot, A. niger was only mildly so. The root-surface mycoflora, therefore, includes fungi which have been reported as the most important in postharvest deterioration of the tubers. The removal of the rhizoplane microflora by surface-sterilization using calcium hypochlorite or Clorox and subsequent incubation in loosely tied polyethylene bags extended the storage life of the tubers considerably.     

Over-production of a glycoside hydrolase family 50 β-agarase from Agarivorans sp. JA-1 in Bacillus subtilis and the whitening effect of its product

The gene for β-agarase of an Agarivorans sp. JA-1 was expressed in Bacillus subtilis strain DB104 for efficient and economical mass-production of the enzyme. We isolated 360 mg protein with a specific activity of 201 U/mg from the culture broth. The efficiency of production was approximately 130-fold higher than that in E. coli. The enzyme produced neoagarohexaose, neoagarotetraose and neoagarobiose from agar. Neoagarooligosaccharides produced by the enzyme had a whitening effect and inhibited tyrosinase activity in the murine melanoma cell line, B16F10. Neoagarooligosaccharides were not cytotoxic to B16F10 or normal cells. β-Agarase could therefore be a good whitening, cosmetic additive.     

Molds on house walls and the effect of their chloroform-extractable metabolites on the respiratory cilia movement of one-day-old chicksin vitro

The ciliostatic activity of the chloroform-extractable endo- and exometabolites of 5 strains of filamentous fungi—Alternaria sp.,Aspergillus glaucus group,Aspergillus versicolor, Cladosporium sphœrospermum, Penicillium sp. andUlocladium sp.—isolated from molded walls of a dwelling—on tracheal cilia from 1-d-old chicksin vitro was evaluated. Endometabolites ofAlternaria sp. andA. versicolor and exometabolites ofUlocladium sp. were the most active, these extracts stopped the ciliary movement within 1 d. The results are discussed in relation to the health status of people living in “moldy” dwellings.     

Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007     

Phylogenomic Analysis of the α Proteasome Gene Family from Early-Diverging Eukaryotes

We employed a phylogenomic approach to study the evolution of α subunits of the proteasome gene family from early diverging eukaryotes. BLAST similarity searches of the Giardia lamblia genome identified all seven α proteasome genes characteristic of eukaryotes from the crown group. In addition, a PCR strategy for the amplification of multiple α subunit sequences generated single α proteasome products for representatives of the Kinetoplastida (Leishmania major), the Parabasalia (Trichomonas vaginalis), and the Microsporidia (Vairimorpha sp., Nosema sp., Endoreticulata sp., and Spraguea lophii). The kinetoplastid Trypanosoma cruzi and the eukaryote crown group Acanthamoeba castellanii yielded two distinct α proteasome genes each. The presence of seven distinct α proteasome genes in G. lamblia, one of the earliest-diverging eukaryotes, indicates that the α proteasome gene family evolved rapidly from a minimum of one gene in Archaea to seven or more in Eukarya. Results from the phylogenomic analysis are consistent with the idea that the Diplomonida (as represented by G. lamblia), the Kinetoplastida, the Parabasalia, and the Microsporidia diverged after the duplication events that originated the α proteasome gene family. A model for the early origin and evolution of the proteasome gene family is presented. Received: 14 February 2000 / Accepted: 14 August 2000     

Antagonistic effects of Streptomyces violaceusniger strain G10 on Fusarium oxysporum f.sp. cubense race 4: Indirect evidence for the role of antibiosis in the antagonistic process

Fusarium oxysporum f.sp. cubense is the causal pathogen of wilt disease of banana. A cost-effective measure of control for this disease is still not available. Streptomyces violaceusniger strain G10 acts as an antifungal agent antagonistic towards many different phytopathogenic fungi, including different pathogenic races of the Fusarium wilt pathogen. In an attempt to understand the mode of action of this antagonist in nature, the interaction between S. violaceusniger strain G10 and F. oxysporum f.sp. cubense was first studied by paired incubation on agar plates. Evidence for the in vitro antibiosis of strain G10 was demonstrated by inhibition zones in the “cross-plug” assay plates. Microscopic observations showed lysis of hyphal ends in the inhibited fungal colonies. Culture of strain G10 in liquid media produces antifungal metabolites, which showed in vitro antagonistic effects against F. oxysporum f.sp. cubense such as swelling, distortion and excessive branching of hyphae, and inhibition of spore germination. An indirect method was used to show that antibiosis is one of the mechanisms of antagonism by which strain G10 acts against F. oxysporun f.sp. cubense in soil. This study suggests the potential of developing strain G10 for the biological control of Fusarium wilt disease of banana. Journal of Industrial Microbiology & Biotechnology (2002) 28, 303–310 DOI: 10.1038/sj/jim/7000247 Received 08 August 2001/ Accepted in revised form 16 February 2002     

Acute phase response in lame cattle with interdigital dermatitis

This study was undertaken to evaluate acute phase response via assessing the concentration of serum sialic acids (total, lipid-bound and protein-bound), inflammatory mediators (IFN-γ and TNF-α) and acute phase proteins [haptoglobin (Hp) and serum amyloid A (SAA)] in lame cattle with interdigital dermatitis. Fifteen hoof scrapings from lame cows were collected from eight commercial dairy farms. As a consequence of the difficulty in culturing and isolation, a PCR technique was used to detect the organism. None of the colonies on enriched blood agar was identified as Fusobacterium necrophorum. Four (26.6%) out of the 15 hoof scrapings examined tested positive for the presence of the lktA gene (402 bp) of F. necrophorum. It seems that culture cannot be considered as the gold standard method for F. necrophorum isolation. Molecular detection is suggested as an alternative method. In the blood serum of different groups of animals (control, lameness and F. necrophorum-positive lameness) Hp, SAA, total sialic acid, lipid-bound sialic acid, and protein-bound sialic acid, and IFN-γ and TNF-α were measured using validated standard procedures. All parameters were significantly higher in the lameness group and the F. necrophorum-positive lameness group compared with the healthy group (P < 0.01 in all cases). Mean SAA concentrations in the lameness group and the F. necrophorum-positive lameness group was relatively 4.6 and 8.0 times higher than the control group. Corresponding measures for Hp indicate a 3.3 times increase in the lameness group compared to the control. In the lameness group, significant associations were observed for Hp with PBSA, SAA with TSA, TSA with PBSA, TSA with LBSA, PBSA with LBSA, and SAA with IFN-γ.     

Aeromycoflora in the Central Milk Dairy of Calcutta, India

Intramural aeromycological survey was performed at the Central Milk Dairy, Calcutta, covering eight locations within the Dairyusing Burkard personal volumetric air sampler. The locations were butter cold storage (−2 °C), cold store (8 °C), packaging section (23 °C), milk processing section (24 °C), reconstituent of skimmed milk (24 °C), quality control lab (25 °C), raw milk reception (28 °C) and loading dock (26 °C). A number of fungal spores, conidia and mycelia were recorded in different rooms: the highest spore quantity was recorded in the packaging section (23 °C) and the minimum at the butter cold store (−2 °C). The dominant spores consisted of Aspergillus niger, A flavus,Cladosporium sp., Fusarium sp., Curvularia sp.,Alternaria sp., Torula sp., Myrotheciumsp., Helminthosporium sp., Periconia sp.,Nigrospora sp. and Pithomyces sp. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of water activity and temperature on in vitro growth of surface cultures of a Phoma sp. and production of the pharmaceutical metabolites, squalestatins S1 and S2

A Phoma sp., known to produce the pharmaceutically active metabolites squalestatin 1 (S1) and squalestatin 2 (S2), was cultured on malt-extract/agar (MEA) over a range of water activities (a _w, 0.995–0.90) and temperatures (10–35 °C) to investigate the influence on growth and metabolite production. Use of the ionic solute NaCl to adjust a _w resulted in significantly lower (P < 0.01) squalestatin yields than when the Phoma sp. was grown on MEA amended with the non-ionic solute glycerol. Water activity and temperature and their interactions were highly significant factors (P < 0.001) affecting growth of the Phoma sp., with optimum conditions of 0.998–0.980 a _w and 25 °C. Squalestatin production was similarly influenced by a _w, temperature, time and their interactions (P < 0.001). S1 and S2 production occurred over a narrower a _w and temperature range than growth, with a slightly lower optimum a _w range of 0.995–0.980 a _w. The optimum temperature for squalestatin production varied from 20 °C (S1) to 25 °C (S2) and yields of S2 were up to 1000 times lower than those of S1. The ratio of S1 and S2 produced by the Phoma sp. was influenced by a _w and temperature, with highest values at 0.99–0.98 a _w, and at 15 °C. Incubation times of 28 days gave highest yields of both S1 and S2. Up to 2000-fold increases in squalestatin yields were measured at optimum environmental conditions, compared to the unmodified MEA. This indicates the need to consider such factors in screening systems used to detect biologically active lead compounds produced by fungi. Received: 2 June 1997 / Received last revision: 6 November 1997 / Accepted: 7 November 1997     

Francisella sp. (Family Francisellaceae) causing mortality in Norwegian cod (Gadus morhua) farming

In 2004, a new disease was detected in cod (Gadus morhua) in western Norway. Affected cod had white granulomas in the visceral organs and skin. A species of Francisella was isolated on blood agar plates from moribund cod. The bacterium could be grown at temperatures ranging from 6 to 22°C, but did not grow at 37°C. Challenge experiments showed that Francisella sp. was the cause for the new disease. The 16S rDNA gene sequence from Francisella sp. showed 99.17% similarity to F. philomiragia, and the 16S–23S ribosomal RNA intergenic spacer (249 nt), shows a similarity with that from Francisella isolated from tilapia and F. tularensis of 96.8 and 35.9%, respectively. The 23S sequence is more similar to F. tularensis, 97.7% (2,862 nt), compared to the tilapia isolate 96.8% (2,131 nt). The partial putative outer membrane protein (FopA) sequence (781 nt) from Francisella sp. shows a similarity with that from F. tularensis and F. philomiragia of 77.3 and 98.2%, respectively. Based on sequence data, culturing temperatures and pathogenicity for cod, it is suggested that this Francisella sp. from cod could be a new species of Francisella, Family Francisellaceae.     

Phenotypic,genomic and phylogenetic characteristics of rhizobia isolated from root nodules of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> (black locust) growing in Poland and Japan

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98–99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771–961 kb megaplasmids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lower Permian Bryozoa from Ellesmere Island (Canada)

A collection of fossils sampled during the 1898–1902 expedition of theFram to the Canadian Arctic Islands includes abundant bryozoans from the Lower Permian (Artinskian) Great Bear Cape Formation of Ellesmere Island. From this material a new genus with one new species —Nansenopora peculiaris n. gen., n. sp. — as well as three new species —Streblotrypella arctica n. sp.,Phragmophera patricki n. sp. andKallodictyon spinatum n. sp. — are described. Furthermore, the speciesUlrichotrypa ramulosa Bassler, 1929 is reported for the first time from the Lower Permian of the Arctic region.     

Toxicity of Hexavalent Chromium and Its Reduction by Bacteria Isolated from Soil Contaminated with Tannery Waste

An Arthrobacter sp. and a Bacillus sp., isolated from a long-term tannery waste contaminated soil, were examined for their tolerance to hexavalent chromium [Cr(VI)] and their ability to reduce Cr(VI) to Cr(III), a detoxification process in cell suspensions and cell extracts. Both bacteria tolerated Cr(VI) at 100 mg/ml on a minimal salts agar medium supplemented with 0.5% glucose, but only Arthrobacter could grow in liquid medium at this concentration. Arthrobacter sp. could reduce Cr(VI) up to 50 μg/ml, while Bacillus sp. was not able to reduce Cr(VI) beyond 20 μg/ml. Arthrobacter sp. was distinctly superior to the Bacillus sp. in terms of their Cr(VI)-reducing ability and resistance to Cr(VI). Assays with permeabilized (treated with toluene or Triton X 100) cells and crude extracts demonstrated that the Cr(VI) reduction was mainly associated with the soluble protein fraction of the cell. Arthrobacter sp. has a great potential for bioremediation of Cr(VI)-containing waste. Received: 13 June 2002 / Accepted: 13 September 2002     

New planthoppers of the tribe Achilini (Homoptera,Fulgoroidea, Achilidae) from Baltic amber

New taxa of Achilini (Achilidae) are described from Baltic amber: Paratesum rasnitsyni gen. et sp. nov., Protomenocria notata gen. et sp. nov., Psycheona variegata gen. et sp. nov., P. striata sp. nov. Protepiptera kaweckii Usinger, 1939 (= Cixidia christinae Lefebvre, Bourgoin et Nel, 2007, syn. nov.) is redescribed with designation of a neotype. “Cixius” testudinarius Germar et Berendt, 1856, “C.” longirostris Germar et Berendt, 1856 and “Oliarus” oligocenus Cockerell, 1910 are transferred to Achilini. A key to the genera of Achilidae known from Baltic amber is provided.     

Monitoring the biofertilizing potential and establishment of inoculated cyanobacteria in soil using physiological and molecular markers

This paper aims to develop methods for quantifying their establishment; using physiological activity (chlorophyll as a growth index and nitrogen-fixing potential as a measure of their biofertilizing capacity), along with evaluation based on DNA fingerprints generated using repeat sequences/palindromes. Time course studies were undertaken in liquid and soil microcosm experiments inoculated with a set of four rhizosphere cyanobacterial strains (BF1 Anabaena sp., BF2 Nostoc sp., BF3 Nostoc sp., BF4 Anabaena sp.). Observations revealed the synergistic effect of three-membered combinations (especially the i.e. BF1 + 2 + 3, 1 + 2 + 4, 1 + 3 + 4) in terms of enhancing chlorophyll and acetylene reducing activity. PCR-based amplification profiles (using short tandemly repetitive repeat (STRR) 1A, STRR_mod, and HIP_AT sequences) proved discriminative in monitoring the presence of the inoculated cyanobacteria in soil microcosm. Future work is in progress to assess the utility of the selected markers/primers in pot experiments, followed by field-level experiments with crop.     

Species of <Emphasis Type="Italic">Neohaliotrema</Emphasis> Yamaguti, 1965 (Monogenea: Ancyrocephalidae) from the pomacentrid <Emphasis Type="Italic">Abudefduf vaigensis</Emphasis> (Quoy &; Gaimard) off Pulau Langkawi,Malaysia, with a revised diagnosis of the genus and a key to its species

Four new and one unidentified species of Neohaliotrema Yamaguti, 1965 were obtained from the gills of the Indo-Pacific sergeant Abudefduf vaigensis (Quoy & Gaimard) off Pulau Langkawi, Malaysia. The five species, N. malayense n. sp., N. bombini n. sp., N. andamanense n. sp., N. parvum n. sp. and an unidentified Neohaliotrema sp. (similar to N. macracanthum Zhukov, 1976), are described and distinguished based mainly on features of the haptor. Species of this genus are divisible into two groups, the ‘maomao group’, with two pairs of morphometrically modified ‘marginal’ hooks and a fenestrated haptor, and the ‘gracile group’, with morphologically similar marginal hooks and an entire haptor. With the exception of N. bombini n. sp., the species described fit within the ‘maomao group’. It is suggested that the more complex Neohaliotrema species of the ‘maomao group’ have modified hooks 1 and 2 on a haptoral ‘isthmus’ between two large apertures, i.e. ‘windows’, whereas the less complex species lacking these features are those of the ‘gracile group’. Neohaliotrema spp. have only a single pair of pigmented eye-spots. A fenestrated haptor is unique to the Neohaliotrema spp. of the ‘maomao group’. The generic diagnosis of Neohaliotrema is amended to include new data and a key to its known species is presented.