First report of branch dieback caused by Neoscytalidium dimidiatum on Styphnolobium japonicum (L.) Schott in China.

Styphnolobium japonicum (L.) Schott (family Fabaceae Juss.) also called pagoda tree, is widely planted in northern China in landscape plantings, for erosion control and forestry. In recent years, symptoms of branch dieback were observed on S. japonicum in the southern part of Xinjiang province, China. From 2019 to 2022, in total ca. 1000 ha area was surveyed in Korla (41.68°N, 86.06°E), Bohu (41.95°N, 86.53°E) and Alaer (41.15°N, 80.29°E). Typical symptoms were observed in 70% of the surveyed branches. To identify the cause, we collected 50 symptomatic branches. Symptoms were initially observed on green current-year twigs, which turned grayish white in color. In the later stages of disease development, a large number of nacked black conidia formed under epidermis of perennial branches, causing visible black protrusions (pycnidia) on branch surface. The disease occurred throughout the entire growing season of S. japonicum. Symptoms also occurred on the inflorescence, fruit, and twigs. In some cases, infection resulted in tree mortality. Isolations were made from the margin between healthy and diseased tissues. Small pieces were excised, surface disinfested (75% ethanol 30 s, 1% NaClO solution 5 mins), cut into pieces (5 to 10 mm2), and incubated on PDA medium at 28℃ for 3 days. A total of 16 isolates (GH01-GH16) with similar colony morphology were obtained. The colonies were initially white, gradually turning to olive-green on the surface and black on the underside after 7 days. Microscopically, the conidia were aseptate, 1-septate, two-septate, and muriform, 2.6-4.5 × 2.9-27.6 µm (n=50). Pycnidia ranged in size from 120.2 to 135.5 × 112.4 to 118.6 µm (n=20). Those morphological characters matched the descriptions of Neoscytalidium dimidiatum (previously N. novaehollandiae) (Alizadeh et al. 2022; Pavlic et al. 2008). For molecular identification, genomic DNA of GH01-GH16 were extracted from fresh mycelia. The internal transcribed spacer (ITS), large subunit ribosomal RNA gene (LSU), and translation elongation factor 1-alpha (EF1-α) gene were amplified using the primer sets ITS1/ITS4 (White 1990), LRoR/LR5 (Vilgalys and Hester 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999). The sequences were deposited in GenBank (accession No. OP379832, OQ096643-OQ096657 for ITS, OP389048, OQ127403-OQ127417 for LSU, and OQ136617, OQ586044-OQ586058 for EF1-α). The ITS sequence had 100% identity (505/505 bp) to MT362600. Similarly, the LSU and EF1-α sequences were found to be identical to MW883823 (100%, 821/821 bp) and KX464763(99%, 256/258 bp), respectively. Pathogenicity was tested on one-year-old healthy S. japonicum seedlings. Spores of representative isolate GH01 were produced on PDA by incubating for 7-days at 28℃. Conidia were washed with sterile water. Five trees were inoculated with 1 × 106 conidia/ml conidial suspensions and five trees were sprayed with sterile water. All trees were covered with plastic bags for 24 h and kept at 25°C in a greenhouse. Signs and symptoms were similar to those observed in field collections one month after inoculation, while no symptoms occurred on the controls. The original fungus was successfully reisolated from the inoculated trees and was identified as N. dimidiatum following the methods described above. N. dimidiatum has been reported in many Asian country such as Malaysia, India, Turkey, and Iran(Akgül et al. 2019; Alizadeh et al. 2022; Khoo et al. 2023; Salunkhe et al. 2023). To our knowledge, this is the first report of N. dimidiatum associated with branch dieback of S. japonicum in China. Our findings have expanded the host range of N. dimidiatum in China and provides a theoretical basis for the diagnosis and treatment of the disease.

Dynamic UAV Phenotyping for Rice Disease Resistance Analysis Based on Multisource Data.

Bacterial blight poses a threat to rice production and food security, which can be controlled through large-scale breeding efforts toward resistant cultivars. Unmanned aerial vehicle (UAV) remote sensing provides an alternative means for the infield phenotype evaluation of crop disease resistance to relatively time-consuming and laborious traditional methods. However, the quality of data acquired by UAV can be affected by several factors such as weather, crop growth period, and geographical location, which can limit their utility for the detection of crop disease and resistant phenotypes. Therefore, a more effective use of UAV data for crop disease phenotype analysis is required. In this paper, we used time series UAV remote sensing data together with accumulated temperature data to train the rice bacterial blight severity evaluation model. The best results obtained with the predictive model showed an R p 2 of 0.86 with an RMSEp of 0.65. Moreover, model updating strategy was used to explore the scalability of the established model in different geographical locations. Twenty percent of transferred data for model training was useful for the evaluation of disease severity over different sites. In addition, the method for phenotypic analysis of rice disease we built here was combined with quantitative trait loci (QTL) analysis to identify resistance QTL in genetic populations at different growth stages. Three new QTLs were identified, and QTLs identified at different growth stages were inconsistent. QTL analysis combined with UAV high-throughput phenotyping provides new ideas for accelerating disease resistance breeding.

Evaluation of rice bacterial blight severity from lab to field with hyperspectral imaging technique.

Hyperspectral imaging technique combined with machine learning is a powerful tool for the evaluation of disease phenotype in rice disease-resistant breeding. However, the current studies are almost carried out in the lab environment, which is difficult to apply to the field environment. In this paper, we used visible/near-infrared hyperspectral images to analysis the severity of rice bacterial blight (BB) and proposed a novel disease index construction strategy (NDSCI) for field application. A designed long short-term memory network with attention mechanism could evaluate the BB severity robustly, and the attention block could filter important wavelengths. Best results were obtained based on the fusion of important wavelengths and color features with an accuracy of 0.94. Then, NSDCI was constructed based on the important wavelength and color feature related to BB severity. The correlation coefficient of NDSCI extended to the field data reached -0.84, showing good scalability. This work overcomes the limitations of environmental conditions and sheds new light on the rapid measurement of phenotype in disease-resistant breeding.

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato.

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

Proteomic approach to understand the molecular physiology of symbiotic interaction between Piriformospora indica and Brassica napus.

Many studies have been now focused on the promising approach of fungal endophytes to protect the plant from nutrient deficiency and environmental stresses along with better development and productivity. Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we used integrated in-depth proteome analyses to characterize the relationship between endophyte Piriformospora indica and Brassica napus plant highlighting its potential involvement in symbiosis and overall growth and development of the plant. An LC-MS/MS based label-free quantitative technique was used to evaluate the differential proteomics under P. indica treatment vs. control plants. In this study, 8,123 proteins were assessed, of which 46 showed significant abundance (34 downregulated and 12 upregulated) under high confidence conditions (p-value ≤ 0.05, fold change ≥2, confidence level 95%). Mapping of identified differentially expressed proteins with bioinformatics tools such as GO and KEGG pathway analysis showed significant enrichment of gene sets involves in metabolic processes, symbiotic signaling, stress/defense responses, energy production, nutrient acquisition, biosynthesis of essential metabolites. These proteins are responsible for root's architectural modification, cell remodeling, and cellular homeostasis during the symbiotic growth phase of plant's life. We tried to enhance our knowledge that how the biological pathways modulate during symbiosis?

Piriformospora indica promotes growth, seed yield and quality of Brassica napus L.

In current scenario, crop productivity is being challenged by decreasing soil fertility. To cope up with this problem, different beneficial microbes are explored to increase the crop productivity with value additions. In this study, Brassica napus L., an important agricultural economic oilseed crop with rich source of nutritive qualities, was interacted with Piriformospora indica, a unique root colonizing fungus with wide host range and multifunctional aspects. The fungus-treated plants showed a significant increase in agronomic parameters with plant biomass, lodging-resistance, early bolting and flowering, oil yield and quality. Nutritional analysis revealed that plants treated by P. indica had reduced erucic acid and glucosinolates contents, and increased the accumulation of N, Ca, Mg, P, K, S, B, Fe and Zn elements. Low erucic acid and glucosinolates contents are important parameters for high quality oil, because oils high in erucic acid and glucosinolates are considered undesirable for human nutrition. Furthermore, the expression profiles of two encoding enzyme genes, Bn-FAE1 and BnECR, which are responsible for regulating erucic acid biosynthesis, were down-regulated at mid- and late- life stages during seeds development in colonized plants. These results demonstrated that P. indica played an important role in enhancing plant growth, rapeseed yield and quality improvement of B. napus.

[Visual Detection Study on Early Bruises of Korla Pear Based on Hyperspectral Imaging Technology].

In this paper, hyperspectral imaging combined with chemometrics was applied for the detection of internal defects of Korla pear. The hyperspectral images covering the spectral range of 380～1 030 nm were acquired for 60 Korla pears before, and seven consecutive days after internal damages were induced by being dropped from a distance of 30 cm. The mean spectrum were computed from region of interests (ROI) of pear in each image, and was preprocessed with wavelet transform for eliminating system noise and external disturbances, and optimizing the spectral identification region (470～963 nm). Based on the preprocessed samples, the support vector machine models were built respectively through the full and feature wavebands selected by the second derivative. The results on testing set demonstrate that both of the two approaches achieved the discrimination accuracy of 93.75%. Furthermore, F-value based method was applied for image analysis to find out the optimal waveband ratio for the visual discrimination of bruises against normal surface. Based on the optimal waveband ratio images, the selective search algorithm was utilized for segmenting bruises from the pear surface, and shows the accurate identification results. Our research revealed that the hyperspectral imaging technique for detecting bruised features in pears is feasible, which could provide a theoretical reference and basis for designing classification system of fruits in further work.

Piriformospora indica confers cadmium tolerance in Nicotiana tabacum.

Piriformospora indica, a root-colonizing endophytic fungus of Sebacinales, promotes plant growth and confers resistance against biotic and abiotic stresses. In order to confirm the influence of P. indica on growth, proline, malondialdehyde (MDA), chlorophyll, and cadmium (Cd) amounts in Nicotiana tabacum under Cd stress, hydroponics, pot and field trials were conducted. The results showed that P. indica can store Cd in plant roots and reduce leaf Cd content, reduce the concentration of MDA, and increase the proline and chlorophyll content and the activities of catalase, peroxidase, and superoxide dismutase under hydroponic Cd stress. RT-PCR analysis showed that the relative expression level of genes Gsh2, TaPCS1, oas1, GPX, and Hsp70 in colonized plants was 4.3, 1.4, 2.9, 1.7, and 6.9 fold higher than in un-colonized plants respectively. Cd exposure significantly reduced un-colonized plants' agronomic traits compared to P. indica-colonized ones. Our results suggested that P. indica can sequester Cd in roots, so that much less cadmium was transported to leaves, and the increased concentrations of antioxidant enzymes, pigments and proline contents, as well as the higher expression of stress-related phytochelatin biosynthesis genes in P. indica-inoculated plants, may also serve to protect N. tabacum plants against oxidative damage, enhancing Cd tolerance.

[Revealing the chemical changes of tea cell wall induced by anthracnose with confocal Raman microscopy].

Healthy tea and tea infected by anthracnose were first studied by confocal Raman microscopy to illustrate chemical changes of cell wall in the present paper. Firstly, Raman spectra of both healthy and infected sample tissues were collected with spatial resolution at micron-level, and ultrastructure of healthy and infected tea cells was got from scanning electron microscope. These results showed that there were significant changes in Raman shift and Raman intensity between healthy and infected cell walls, indicating that great differences occurred in chemical compositions of cell walls between healthy and infected samples. In details, intensities at many Raman bands which were closely associated with cellulose, pectin, esters were reduced after infection, revealing that the content of chemical compounds such as cellulose, pectin, esters was decreased after infection. Subsequently, chemical imaging of both healthy and infected tea cell walls were realized based on Raman fingerprint spectra of cellulose and microscopic spatial structure. It was found that not only the content of cellulose was reduced greatly after infection, but also the ordered structure of cellulose was destroyed by anthracnose infection. Thus, confocal Raman microscopy was shown to be a powerful tool to detect the chemical changes in cell wall of tea caused by anthracnose without any chemical treatment or staining. This research firstly applied confocal Raman microscopy in phytopathology for the study of interactive relationship between host and pathogen, and it will also open a new way for intensive study of host-pathogen at cellular level.

The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses.

BACKGROUND: Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years. RESULTS: Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host. CONCLUSIONS: We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.

Isolation and characterization of bacteria from midgut of the rice water weevil (Coleoptera: Curculionidae).

Gut bacteria are known to play important and often essential roles in the biology of insects. Theoretically, they can be genetically manipulated, then reintroduced into insects to negatively modify specific biological features. The weevil superfamily Curculionoidea is one of the most species-rich and successful animal groups on earth, but currently the overall knowledge of the bacterial communities in weevils and their associations with hosts is still limited. In this study, we isolated and characterized the bacteria in the midgut of an invasive weevil, Lissorhoptrus oryzophilus Kuschel, by culturing methods. Female adults of this weevil were collected from four different geographic regions of the United States and mainland China. Sequencing of the bacterial 16S rRNA amplicons demonstrated that the major culturable gut bacteria of rice water weevil are γ-proteobacteria and Bacilli. The gut bacterial composition differs among regions, with many of the bacteria isolated from only a single region while several were detected from more than one region. Overall, the diversity of gut bacteria in rice water weevil is relatively low. The possible origins of certain bacteria are discussed in relation to the weevil, rice plant, and bacteria.

Growth promotion of Chinese cabbage and Arabidopsis by Piriformospora indica is not stimulated by mycelium-synthesized auxin.

Piriformospora indica, an endophytic fungus of the order Sebacinales, interacts with the roots of a large variety of plant species. We compared the interaction of this fungus with Chinese cabbage (Brassica campestris subsp. chinensis) and Arabidopsis seedlings. The development of shoots and roots of Chinese cabbage seedlings was strongly promoted by P. indica and the fresh weight of the seedlings increased approximately twofold. The strong stimulation of root hair development resulted in a bushy root phenotype. The auxin level in the infected Chinese cabbage roots was twofold higher compared with the uncolonized controls. Three classes of auxin-related genes, which were upregulated by P. indica in Chinese cabbage roots, were isolated from a double-subtractive expressed sequence tag library: genes for proteins related to cell wall acidification, intercellular auxin transport carrier proteins such as AUX1, and auxin signal proteins. Overexpression of B. campestris BcAUX1 in Arabidopsis strongly promoted growth and biomass production of Arabidopsis seedlings and plants; the roots were highly branched but not bushy when compared with colonized Chinese cabbage roots. This suggests that BcAUX1 is a target of P. indica in Chinese cabbage. P. indica also promoted growth of Arabidopsis seedlings but the auxin levels were not higher and auxin genes were not upregulated, implying that auxin signaling is a more important target of P. indica in Chinese cabbage than in Arabidopsis. The fungus also stimulated growth of Arabidopsis aux1 and aux1/axr4 and rhd6 seedlings. Furthermore, a component in an exudate fraction from P. indica but not auxin stimulated growth of Chinese cabbage and Arabidopsis seedlings. We propose that activation of auxin biosynthesis and signaling in the roots might be the cause for the P. indica-mediated growth phenotype in Chinese cabbage.

Effect of water activity on stress tolerance and biocontrol activity in antagonistic yeast Rhodosporidium paludigenum.

The aims of this study were to optimize the salt-adaptation conditions of the marine antagonistic yeast Rhodosporidium paludigenum and investigate the biocontrol activity of salt-induced cell suspensions of R. paludigenum on postharvest pathogens in fruits. Low water activity (a(w)=0.98, 0.97, 0.96, and 0.95) inhibited the growth of R. paludigenum in nutrient yeast dextrose broth, but the yeast grew better in the medium modified with NaCl solute than other nonionic solutes. R. paludigenum grown in 6.6% NaCl-modified medium had higher viabilities (92.1%) at low water activity (a(w)=0.95) than control (81.1%) after 48h incubation. The salt-adapted R. paludigenum also showed better viability than the un-adapted cells after being frozen, which may be related to the accumulation of intracellular trehalose. Moreover, the best biocontrol inhibition in pears and Chinese winter jujubes was obtained when R. paludigenum was grown in NaCl-modified medium. Therefore, this study implies that improving physiological inducement methods may be a promising strategy for accelerating commercialization of biocontrol agents.

[Study on the early detection of Sclerotinia of Brassica napus based on combinational-stimulated bands].

The combinational-stimulated bands were used to develop linear and nonlinear calibrations for the early detection of sclerotinia of oilseed rape (Brassica napus L.). Eighty healthy and 100 Sclerotinia leaf samples were scanned, and different preprocessing methods combined with successive projections algorithm (SPA) were applied to develop partial least squares (PLS) discriminant models, multiple linear regression (MLR) and least squares-support vector machine (LS-SVM) models. The results indicated that the optimal full-spectrum PLS model was achieved by direct orthogonal signal correction (DOSC), then De-trending and Raw spectra with correct recognition ratio of 100%, 95.7% and 95.7%, respectively. When using combinational-stimulated bands, the optimal linear models were SPA-MLR (DOSC) and SPA-PLS (DOSC) with correct recognition ratio of 100%. All SPA-LSSVM models using DOSC, De-trending and Raw spectra achieved perfect results with recognition of 100%. The overall results demonstrated that it was feasible to use combinational-stimulated bands for the early detection of Sclerotinia of oilseed rape, and DOSC-SPA was a powerful way for informative wavelength selection. This method supplied a new approach to the early detection and portable monitoring instrument of sclerotinia.

Piriformospora indica confers drought tolerance in Chinese cabbage leaves by stimulating antioxidant enzymes, the expression of drought-related genes and the plastid-localized CAS protein.

Piriformospora indica, a root-colonizing endophytic fungus of Sebacinales, promotes plant growth and confers resistance against biotic and abiotic stress. The fungus strongly colonizes the roots of Chinese cabbage, promotes root and shoot growth, and promotes lateral root formation. When colonized plants were exposed to polyethylene glycol to mimic drought stress, the activities of peroxidases, catalases and superoxide dismutases in the leaves were upregulated within 24h. The fungus retarded the drought-induced decline in the photosynthetic efficiency and the degradation of chlorophylls and thylakoid proteins. The expression levels of the drought-related genes DREB2A, CBL1, ANAC072 and RD29A were upregulated in the drought-stressed leaves of colonized plants. Furthermore, the CAS mRNA level for the thylakoid membrane associated Ca(2+)-sensing regulator and the amount of the CAS protein increased. We conclude that antioxidant enzyme activities, drought-related genes and CAS are three crucial targets of P. indica in Chinese cabbage leaves during the establishment of drought tolerance. P. indica-colonized Chinese cabbage provides a good model system to study root-to-shoot communication.

Expression of BvGLP-1 encoding a germin-like protein from sugar beet in Arabidopsis thaliana leads to resistance against phytopathogenic fungi.

Nematode (Heterodera schachtii) resistance in sugar beet (Beta vulgaris) is controlled by a single dominant resistance gene, Hs1(pro-1). BvGLP-1 was cloned from resistant sugar beet. The BvGLP-1 messenger (m)RNA is highly upregulated in the resistant plants after nematode infection, suggesting its role in the Hs1(pro-1) mediated resistance. BvGLP-1 exhibits sequence homology to a set of plant germin-like proteins (GLP), from which several have proved to be functional in plant basal or defense resistance against fungal pathogens. To test whether BvGLP-1 is also involved in the plant-fungus interaction, we transferred BvGLP-1 into Arabidopsis and challenged the transgenic plants with the pathogenic fungi Verticillium longisporum and Rhizoctonia solani as well as with the beneficial endophytic fungus Piriformospora indica. The expression of BvGLP-1 in Arabidopsis elevated the H(2)O(2) content and conferred significant resistance to V. longisporum and R. solani but did not affect the beneficial interaction with P. indica in seedlings. Microscopic observations revealed a dramatic reduction in the amount of hyphae of the pathogenic fungi on the root surface as well as of fungal mycelium developed inside the roots of transgenic Arabidopsis compared with wild-type plants. Molecular analysis demonstrated that the BvGLP-1 expression in Arabidopsis constitutively activates the expression of a subset of plant defense-related proteins such as PR-1 to PR-4 and PDF1.2 but not PDF2.1 and PDF2.3. In contrast, the PDF2.1 mRNA level was downregulated. These data suggest an important role of BvGLP-1 in establishment of plant defense responses, which follow specific signaling routes that diverge from those induced by the beneficial fungus.

[Identification and classification of rice leaf blast based on multi-spectral imaging sensor].

Site-specific variable pesticide application is one of the major precision crop production management operations. Rice blast is a severe threat for rice production. Traditional chemistry methods can do the accurate crop disease identification, however they are time-consuming, require being executed by professionals and are of high cost. Crop disease identification and classification by human sight need special crop protection knowledge, and is low efficient. To obtain fast, reliable, accurate rice blast disease information is essential for achieving effective site-specific pesticide applications and crop management. The present paper describes a multi-spectral leaf blast identification and classification image sensor, which uses three channels of crop leaf and canopy images. The objective of this work was to develop and evaluate an algorithm under simplified lighting conditions for identifying damaged rice plants by the leaf blast using digital color images. Based on the results obtained from this study, the seed blast identification accuracy can be achieved at 95%, and the leaf blast identification accuracy can be achieved at 90% during the rice growing season. Thus it can be concluded that multi-spectral camera can provide sufficient information to perform reasonable rice leaf blast estimation.

Molecular identification of cDNA, immunolocalization, and expression of a putative odorant-binding protein from an Asian honey bee, Apis cerana cerana.

An odorant-binding protein cDNA (Acer-ASP2) was cloned and characterized from antennae of adult workers of an Asian honey bee, Apis cerana cerana F. (Hymenoptera: Apidae). The full-length open reading frame of Acer-ASP2 cDNA was 429 bp, encoding 142 amino acids. Protein signature analyses revealed that it contained six conserved cysteines with an N-terminal signal sequence of 19 amino acids. The deduced protein sequence shares high homology with Amel-ASP2 from the honey bee, Apis mellifera L., and low similarity with odorant-binding proteins from other species of insects. Immunocytochemical localization showed that Acer-ASP2 was concentrated in the lymph of olfactory sensilla, such as sensilla placodea and sensilla trichodea A. Real-time polymerase chain reaction of Acer-ASP2 transcripts showed that Acer-ASP2 was expressed on antennae but not in other general anatomical regions of the body. Temporally, Acer-ASP2 was expressed at a relatively high level in adults during two periods (9 and 27 vs. 1, 15, and 30 days). This timing is correlated with the production of beeswax and searching behavior for nectar/pollen, respectively. Thus, Acer-ASP2 is a species-specific gene that we propose to be involved in the acquisition of odorant molecules from nectar, pollen, and other general odorant sources.

Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis.

A new feather-degrading bacterium was isolated from a local feather waste site and identified as Bacillus subtilis based on morphological, physiochemical, and phylogenetic characteristics. Screening for mutants with elevated keratinolytic activity using N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis resulted in a mutant strain KD-N2 producing keratinolytic activity about 2.5 times that of the wild-type strain. The mutant strain produced inducible keratinase in different substrates of feathers, hair, wool and silk under submerged cultivation. Scanning electron microscopy studies showed the degradation of feathers, hair and silk by the keratinase. The optimal conditions for keratinase production include initial pH of 7.5, inoculum size of 2% (v/v), age of inoculum of 16 h, and cultivation at 23 degrees C. The maximum keratinolytic activity of KD-N2 was achieved after 30 h. Essential amino acids like threonine, valine, methionine as well as ammonia were produced when feathers were used as substrates. Strain KD-N2, therefore, shows great promise of finding potential applications in keratin hydrolysis and keratinase production.