Contaminated bacterial effects and qPCR application to monitor a specific bacterium in <Emphasis Type="Italic">Chlorella</Emphasis> sp. KR-1 culture

To investigate the effects of bacteria contaminated in microalgal cultivation, several bacteria were isolated from four photobioreactors for Chlorella sp. KR-1 culture. A total of twenty-one bacterial strains isolated from the reactors and identified by 16S rRNA gene sequencing. Six bacteria, which were found from more than two reactors of the four photobioreactors, were introduced into co-culturing experiments with Chlorella sp. KR-1. Then, the bacterial influences on the productivity of microalgal biomass and lipids were assessed in the photoautotrophic- and mixotrophic microalgal cultivation by comparing them with axenic culture of Chlorella sp. KR-1. The results showed that both biomass and lipid production were significantly enhanced under mixotrophic conditions compared to photoautotropic conditions. However, an excess ratio (more than 10%) of bacterial cells to microalgal cells at the initial stage of mixotrophic cultivation has limited the growth of Chlorella sp. KR-1 because of the relatively fast growth of bacteria, especially under mixotrophic conditions. Moreover, it was proven that the strong biofilm formability of Sphingomonas sp. MB6 is the responsible strain to cause the biomass aggregation observed during the early stage of co-culture. The high abundance of Sphingomonas sp. MB6 during early cultivation period shown by qPCR results was also well corresponded with the period shown a strong biofilm formation, which suggested the applicability of qPCR to monitor a specific bacterial group in a microalgal culture.     

Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, <Emphasis Type="Italic">Chlorella</Emphasis> sp. Isolated from Dairy Wastewater

The present study focused on cost-effective production of microalgal biomass and lipid production on dairy effluent. The novel microalga, Chlorella sp. isolated from the dairy effluent showed high growth and lipid production on the undiluted and two-fold diluted dairy effluent which were four to five times higher than those of Chlorella vulgaris (control). The high growth of Chlorella sp. was thought to be possibly due to its heterotrophic growth capacity, high turbidity, COD, nutrients and trace elements. In contrast, C. vulgaris showed poor heterotrophic and photoautotrophic growth under the highly turbid conditions of dairy effluent. Both Chlorella sp. and C. vulgaris showed similar total FAME (mg FAME/g algal cells). The fatty acid composition analysis revealed that both Chlorella sp. and C. vulgaris possessed major C18 and C20 fatty acids which will be used for biodiesel production. Overall, the novel microalga, Chlorella sp. isolated from the dairy effluent showed high potential for cost-effective algal cultivation and lipid production on dairy effluent without any modification of process.     

Effects of <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> extract on growth and antioxidant defense systems of two freshwater microalgae

Microalgae in genus Chlorella and Scenedesmus are common in aquatic ecosystems and are widely used for various studies on algal growth and applications. Macroalgae may play an important role for control of microalgal growth, attributable to their rich content of bioactive compounds. In this study, the brown seaweed Ascophyllum nodosum was extracted with 70% acetone and the extract was used to treat the green microalgae, Chlorella vulgaris and Scenedesmus sp. Cell density and chlorophyll a concentration were used as growth indexes to evaluate the effects of A. nodosum extract (ANE) on the microalgae. The ANE with concentrations > 1% exhibited significant capability of inhibition of the growth of microalgae by over 80%. On the contrary, 1% ANE caused varying degrees of acceleration of cell proliferation and chlorophyll a synthesis in C. vulgaris and Scenedesmus sp., respectively. Analysis of antioxidant activities of the enzymes superoxide dismutase (SOD) and catalase (CAT) revealed the impact of ANE on the antioxidant defense system of the microalgae. The SOD and CAT activities were significantly depressed by high concentrations (> 2%) ANE, while a slight increase of the enzyme activities was observed with 1% ANE at the early period, which could be correlated to the growth response. Therefore, the mechanism of microalgae control could be related to the interaction between the ANE and the antioxidant defense systems. Phlorotannins are proposed as the principal algistatic components in the ANE which could be utilized in controlling microalgae growth.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Productivity and biochemical composition of <Emphasis Type="Italic">Tetradesmus obliquus</Emphasis> and <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>: effects of different cultivation approaches

The present work evaluated biomass productivity, carbon dioxide fixation rate, and biochemical composition of two microalgal species, Phaeodactylum tricornutum (Bacillariophyta) and Tetradesmus obliquus (Chlorophyta), cultivated indoors in high-technology photobioreactors (HT-PBR) and outdoors both in pilot ponds and low-technology photobioreactors in a greenhouse in southern Italy. Microalgae were grown in standard media, under nitrogen starvation, and in two liquid digestates obtained from anaerobic digestion of agro-zootechnical and vegetable biomass. P. tricornutum, cultivated in semi-continuous mode in indoor HT-PBRs with standard medium, showed a biomass productivity of 21.0?±?2.3 g m^?2 d^?1. Applying nitrogen starvation, the lipid productivity increased from 2.3 up to 4.5?±?0.5 g m^?2 d^?1, with a 24 % decrease of biomass productivity. For T. obliquus, a biomass productivity of 9.1?±?0.9 g m^?2 d^?1 in indoor HT-PBR was obtained using standard medium. Applying liquid digestates as fertilizers in open ponds, T. obliquus gave a biomass productivity (10.8?±?2.0 g m^?2 d^?1) not statistically different from complete medium such as P. tricornutum (6.5?±?2.2 g m^?2 d^?1). The biochemical data showed that the fatty acid composition of the microalgal biomass was affected by the different cultivation conditions for both microalgae. In conclusion, it was found that the microalgal productivity in standard medium was about doubled in HT-PBR compared to open ponds for P. tricornutum and was about 20 % higher for T. obliquus.     

Reduced gene expression at the branch point of chlorophyll and heme biosynthesis in Arctic <Emphasis Type="Italic">Chlorella</Emphasis> ArM0029B

The gene expression at the branch point of chlorophyll and heme synthesis in the model microalga, Chlamydomonas reinhardtii, is different from that of higher plants. Another green alga, Arctic Chlorella, was recently isolated from Arctic sea ice and may be a promising candidate for a biofuel. To understand the chlorophyll metabolic pathway and relevant nuclear gene expression in Chlorella sp., we characterized chlorophyll-deficient mutants of the Arctic Chlorella sp. ArM0029B. First, we characterized the chlorophyll and heme biosynthetic pathways based on genes identified by bioinformatics analysis of the genome of Arctic Chlorella sp. ArM0029B. Then, we isolated and analyzed nine chlorophyll-deficient mutants that showed reduced expression of the ChlM gene, which encodes Mg-protoporphyrin methyltransferase. Expression of 5-amino levulinic acid dehydratase (encoded by ALAD) and glutamyl-tRNA reductase (encoded by HemA) was reduced in all nine independent mutants compared to wild type. These results indicated that Arctic Chlorella ArM0029B may have a regulatory mechanism of gene expression at earlier steps of the Mg-porphyrin branch that is more similar to higher plants than to the microalga C. reinhardtii. This study provides useful insight into the regulation of porphyrin precursor formation in Chlorella and related microalgae.     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.     

Effect of microalgae hydrolysate foliar application (<Emphasis Type="Italic">Arthrospira platensis</Emphasis> and <Emphasis Type="Italic">Scenedesmus</Emphasis> sp.) on <Emphasis Type="Italic">Petunia x hybrida</Emphasis> growth

In horticultural practice accelerated plant development and particularly earlier flowering, has been reported with microalgae applications. Therefore, the objective of this work was to study the effects of foliar spraying with Scenedesmus sp. and Arthrospira platensis hydrolysates on Petunia x hybrida plant development and leaf nutrient status. Three treatments were tested: T1 (foliar application with water, the control), T2 (foliar application with Arthrospira), and T3 (foliar application with Scenedesmus). Foliar spraying was applied five times (0, 14, 28, 35, and 42 days after transplanting). The concentration of both microalgae was 10 g L^?1. At the end of the trial biometric parameters and nutrient concentration in photosynthetic organs (the leaves) were measured. The results of this assay show that foliar application of Scenedesmus accelerated plant development in terms of higher rates of root growth, leaf and shoot development, and earliness of flowering. Arthrospira enhanced the root dry matter, the number of flowers per plant, and the water content. Nevertheless, a reduction was found in the conductive tissue (stem + petiole) dry weight with Arthrospira compared with Scenedesmus and the control. The results also show that microalgae hydrolysate supply can improve the plant nutrient status. Based on these results, it is advisable to use Scenedesmus hydrolysates in foliar applications to increase the blooming of Petunia x hybrida.     

The eurythermal adaptivity and temperature tolerance of a newly isolated psychrotolerant Arctic <Emphasis Type="Italic">Chlorella</Emphasis> sp.

A new strain of Chlorella sp. (Chlorella-Arc), isolated from Arctic glacier melt water, was found to have high specific growth rates (μ) between 3 and 27 °C, with a maximum specific growth rate of 0.85 day^?1 at 15 °C, indicating that this strain was a eurythermal strain with a broad temperature tolerance range. To understand its acclimation strategies to low and high temperatures, the physiological and biochemical responses of the Chlorella-Arc to temperature were studied and compared with those of a temperate Chlorella pyrenoidosa strain (Chlorella-Temp). As indicated by declining F _v/F _m, photoinhibition occurred in Chlorella-Arc at low temperature. However, Chlorella-Arc reduced the size of the light-harvesting complex (LHC) to alleviate photoinhibition, as indicated by an increasing Chl a/b ratio with decreasing temperatures. Interestingly, Chlorella-Arc tended to secrete soluble sugar into the culture medium with increasing temperature, while its intracellular soluble sugar content did not vary with temperature changes, indicating that the algal cells might suffer from osmotic stress at high temperature, which could be adjusted by excretion of soluble sugar. Chlorella-Arc accumulated protein and lipids under lower temperatures (<15 °C), and its metabolism switched to synthesis of soluble sugar as temperatures rose. This reflects a flexible ability of Chlorella-Arc to regulate carbon and energy distribution when exposed to wide temperature shifts. More saturated fatty acids (SFA) in Chlorella-Arc than Chlorella-Temp also might serve as the energy source for growth in the cold and contribute to its cold tolerance.     

Identification of <Emphasis Type="Italic">Pm58</Emphasis> from <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Key message

A novel powdery mildew-resistance gene, designated Pm58, was introgressed directly from Aegilops tauschii to hexaploid wheat, mapped to chromosome 2DS, and confirmed to be effective under field conditions. Selectable KASP? markers were developed for MAS.

Abstract

Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) remains a significant threat to wheat (Triticum aestivum L.) production. The rapid breakdown of race-specific resistance to Bgt reinforces the need to identify novel sources of resistance. The d-genome species, Aegilops tauschii, is an excellent source of disease resistance that is transferrable to T. aestivum. The powdery mildew-resistant Ae. tauschii accession TA1662 (2n?=?2x?=?DD) was crossed directly with the susceptible hard white wheat line KS05HW14 (2n?=?6x?=?AABBDD) followed by backcrossing to develop a population of 96 BC₂F₄ introgression lines (ILs). Genotyping-by-sequencing was used to develop a genome-wide genetic map that was anchored to the Ae. tauschii reference genome. A detached-leaf Bgt assay was used to screen BC₂F_4:6 ILs, and resistance was found to segregate as a single locus (χ?=?2.0, P value?=?0.157). The resistance gene, referred to as Pm58, mapped to chromosome 2DS. Pm58 was evaluated under field conditions in replicated trials in 2015 and 2016. In both years, a single QTL spanning the Pm58 locus was identified that reduced powdery mildew severity and explained 21% of field variation (P value?<?0.01). KASP? assays were developed from closely linked GBS-SNP markers, a refined genetic map was developed, and four markers that cosegregate with Pm58 were identified. This novel source of powdery mildew-resistance and closely linked genetic markers will support efforts to develop wheat varieties with powdery mildew resistance.

     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

Overexpression of <Emphasis Type="Italic">flv3</Emphasis> improves photosynthesis in the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6803 by enhancement of alternative electron flow

Background

To ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.

Results

In the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O₂ photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O₂ evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo¹³C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.

Conclusions

Overexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO₂ incorporation is enhanced by the flv3 overexpression. Increase in O₂ evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

High pH-induced flocculation of marine <Emphasis Type="Italic">Chlorella</Emphasis> sp. for biofuel production

Microalgae are being considered as a promising raw material for biofuel production. However, rapid, efficient, and economic technologies for harvesting microalgae are essential for successful applications. In this study, the high–pH-induced flocculation method was applied to harvest marine Chlorella sp. strains. These algae could be concentrated up to approximately 20-fold by increasing pH using NaOH, with a flocculation efficiency of 90 %. When NaOH dosage was low (1 or 3 mM), the flocculation efficiency decreased considerably with the increase of biomass concentration. At higher NaOH dosage tested (5 or 7 mM), flocculation occurred quickly and efficiently, which tended to be independent of biomass concentration. In larger volumes, all strains were flocculated with similar efficiencies (approximately 90 %) after adding 5 mM NaOH. After flocculation, the flocculated algae cells could be re-cultured as inoculum, and the growth yields in flocculated medium were slightly higher than those from fresh medium. Additionally, for each strain, there were no significant differences in lipid extraction yield and fatty acid composition according to different harvesting methods. These results showed that the high–pH-induced flocculation method could be used to harvest marine Chlorella sp. for biofuel production successfully.     

Growth and photosynthetic activity of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> biofilms

Botryococcus braunii is a green microalga capable of producing large amounts of external long-chain hydrocarbons suitable as a source of biofuel. There have been several studies indicating that cultures of B. braunii can reduce the energy and water requirement for mass biofuel production, especially if non-destructive extraction methods for milking hydrocarbons are used. Growing microalgae as a raw material for biofuel using conventional liquid-based cultivation (i.e., raceway ponds) has yet to be shown to be economically successful. An alternative solid growth (biofilm) cultivation method can markedly reduce the energy requirements and costs associated with the harvesting and dewatering processes. We evaluated the growth of biofilms of several strains of B. braunii (from races A, B, L and S) and found that three of the four tested races successfully grew to stationary phase in 10 weeks with no contamination. Among all races, B. braunii BOT22 (race B) reached the highest biomass and lipid yields (3.80 mg dry weight cm^?2 day^?1 and 1.11 mg dry weight cm^?2). Irrespective of the race, almost all photosynthetic parameters (F _V /F ₀ , PI_ABS and the OJIP curve) showed that the biofilm cultures were more stressed during lag and stationary phases than in logarithmic phase. We also studied the Botryococcus biofilm profiles using confocal microscopy and found that this method is suitable for estimating the overall biomass yield when compared with gravimetric measurement. In conclusion, the growth characteristics (biomass and lipid) and photosynthetic performance of all races indicated that B. braunii BOT22 is the most promising strain for biofilm cultivation.     

Enhanced succinic acid productivity by expression of <Emphasis Type="Italic">mgtCB</Emphasis> gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> mutant

In this study, a novel engineering Escherichia coli strain (CBMG111) with the expression of mgtCB gene was constructed for the enhanced fermentative production of succinic acid by utilizing the synergetic effect of mgtC gene to improve the growth of strains at the environment of low Mg²⁺ concentration and mgtB to enhance the transport of Mg²⁺ into cells. After the effect of the expression of the individual genes (mgtA, mgtB, mgtC) on the growth of E. coli was clarified, the fermentative production of succinic acid by CBMG111 was studied with the low-price mixture of Mg(OH)₂ and NH₃·H₂O as the alkaline neutralizer and the biomass hydrolysates as the carbon sources, which demonstrated that the expression of mgtCB gene can significantly increase the productivity of succinic acid (2.97 g L^?1 h^?1) compared with that by using the engineering strain with the overexpression of mgtA gene.     

Identification of 21 novel <Emphasis Type="Italic">S-RNase</Emphasis> alleles and determination of <Emphasis Type="Italic">S</Emphasis>-genotypes in 66 loquat (<Emphasis Type="Italic">Eriobotrya</Emphasis>) accessions

As observed in other self-incompatible species in the Pyrinae subtribe, loquat (Eriobotrya japonica) demonstrates gametophytic self-incompatibility that is controlled by the S-locus, which encodes a polymorphic stylar ribonuclease (S-RNase). This allows the female reproductive organ (style) to recognize and reject the pollen from individuals with the same S-alleles, but allows the pollen from individuals with different S-alleles to effect fertilization. The S-genotype is therefore an important consideration in breeding strategies and orchard management. In an attempt to optimize the selection of parental lines in loquat production, the S-RNase alleles of 35 loquat cultivars and their 26 progeny, as well as five wild loquat species, were identified and characterized in this study. The best pollinizer cultivar combinations were also explored. A total of 28 S-alleles were detected, 21 of which constituted novel S-RNase alleles. The S-haplotypes S₂ and S₆ were the most frequent, followed by S ₂₉ , S ₃₁ , S ₅ , S ₂₄ , S ₂₈ , S ₃₃ , S ₃₄ , S ₃₂ , and S ₁₅ , while the rare alleles S ₁ , S ₉ , S ₁₄ , S ₁₆ , S ₁₇ , S ₁₈ , S ₁₉ , S ₂₀ , S ₂₁ , S ₂₂ , S ₂₃ , S ₂₇ , and S ₃₅ were only observed in one of the accessions tested. Moreover, the S-genotypes of five wild loquat species (E. prinoides, E. bengalensis, E. prinoides var. dadunensis, E. deflexa, and E. japonica) are reported here for the first time. The results will not only facilitate the selection of suitable pollinators for optimal orchard management, but could also encourage the crossbreeding of wild loquat species to enhance the genetic diversity of loquat cultivars.     

New species of the tenebrionid-beetle genera <Emphasis Type="Italic">Tagonoides</Emphasis> Fairm., <Emphasis Type="Italic">Gnaptorina</Emphasis> Rtt. and <Emphasis Type="Italic">Agnaptoria</Emphasis> Rtt. (Coleoptera,Tenebrionidae) from China

Six new species Tagonoides belousovi sp. n., T. yunnana sp. n., Gnaptorina australis sp. n., Agnaptoria elongata sp. n. (Yunnan), A. lecta sp. n., and A. ruida sp. n. (Sichuan), and a new subspecies Gnaptorina potanini minxiana subsp. n. are described from China. Morphological adaptations of tenebrionids of the tribe Blaptini associated with phytophagous habit are discussed.     

New <Emphasis Type="Italic">Penicillium</Emphasis> and <Emphasis Type="Italic">Talaromyces</Emphasis> species from honey,pollen and nests of stingless bees

Penicillium and Talaromyces species have a worldwide distribution and are isolated from various materials and hosts, including insects and their substrates. The aim of this study was to characterize the Penicillium and Talaromyces species obtained during a survey of honey, pollen and the inside of nests of Melipona scutellaris. A total of 100 isolates were obtained during the survey and 82% of those strains belonged to Penicillium and 18% to Talaromyces. Identification of these isolates was performed based on phenotypic characters and β-tubulin and ITS sequencing. Twenty-one species were identified in Penicillium and six in Talaromyces, including seven new species. These new species were studied in detail using a polyphasic approach combining phenotypic, molecular and extrolite data. The four new Penicillium species belong to sections Sclerotiora (Penicillium fernandesiae sp. nov., Penicillium mellis sp. nov., Penicillium meliponae sp. nov.) and Gracilenta (Penicillium apimei sp. nov.) and the three new Talaromyces species to sections Helici (Talaromyces pigmentosus sp. nov.), Talaromyces (Talaromyces mycothecae sp. nov.) and Trachyspermi (Talaromyces brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated here.