Spore Culture Research Articles

First report of Fusarium solani Species Complex Causing Root Rot of Loquat (Eriobotrya japonica) in China.

Loquat (Eriobotrya japonica), a native fruit tree to China, is a popular edible fruit with medicinal properties (Badenes et al. 2013). A 2016-2019 field survey of ~13,000 loquat trees in two orchards in Chongqing and Fujian provinces showed about 5 to 10% root rot disease incidence. The disease symptoms included leaf yellowing, wilting, rotting of main root, and cracking of lateral roots, eventually leading to defoliation and death. To determine the causative agent, diseased roots from six trees were collected, washed in tap water, cut into 2-3 mm pieces, and disinfected for 3 min in 75% (v/v) EtOH. After rinsing in sterilized water, the root pieces were soaked in 10% NaClO (w/v) for 5-10 min, rinsed thrice in sterile water, and plated on potato dextrose agar (PDA). After 7 days of incubation at 25°C, individual spores were collected from the fungal colonies and replated. Single spore cultures growing on PDA gave rise to woolly-cottony, cream-white colored aerial mycelium and a yellowish pigmented mycelium. The average colony growth rate was 8.6 mm day-1 (n=3). Microscopic observation of the mycelium revealed septate and hyaline hyphae and long cylindrical monophialides. Macroconidia were moderately curved, stout, 3-4 septate, measuring 20.79-48.70 μm × 4.16-10.14 μm (n=50). Microconidia produced from long phialides were kidney-shaped, 0-2 septate, and 5.72-17.28 μm × 2.29-6.51 μm (n=50) in size. The mycelial characteristics and reproductive structures of the isolates fit the morphological description of Fusarium sp. (Summerell et al. 2003). To confirm this identification, translation elongation factor (EF-1α) and RNA polymerase I beta subunit (RPB1) and RNA polymerase II beta subunit (RPB2) regions of the genome were PCR amplified from 3 separate isolates (R2, R4 and R5) using EF1/ EF2, RPB1-Fa/G2R, RPB2-5f2/7cR & RPB2-7cF/11aR primer pairs (O'Donnell et al. 2010) and sequenced. BLASTn comparison of the EF-1α (MT976167), RPB1 (MT967271) and RPB2 (MW233052) regions from isolate R4 showed 99% identity with the EF-1α (GU170620, 675/676 bp), RPB1 (KC808270, 1543/1545 bp) and RPB2 (MK4419902, 1637/1638 bp) sequences of Fusarium solani species complex (FSSC) in GenBank database. The same species level identification was also found using FUSARIUM-ID and FUSARIUM-MLDT databases. Two-year-old seedlings (n=3) of two different cultivars, 'Hunanzaoshu' and 'Huabai No. 1', growing in pots indoors at 25-27 °C were inoculated by drenching the soil with a conidial suspension of isolate R4 (40 mL, 106 conidia mL-1 obtained from 6-10 day old cultures). Control plants (n=3) were inoculated with sterilized water. At 20 days after inoculation (DAI) the leaves of inoculated plants became chlorotic and wilted, defoliated over time, and by 53 DAI 91.67% of plants died. The taproot and lateral roots of inoculated plants appeared brown to black in color and most lateral roots died and decomposed at 53 DAI, whereas the control plant roots remained healthy. All control plants remained symptomless. Based on morphological and molecular characters (TEF-1, RPB1 and RPB2), the re-isolated pathogen from diseased plants was identical to the R4 isolate used for inoculation and the disease assays were repeated thrice. FSSC was recently reported to cause fruit rot disease on loquat in Pakistan (Abbas et al. 2017). Identifying Fusarium solani species complex as a disease agent in Chinese loquat will assist in future development of improved germplasm for this important worldwide tree crop.

Fusarium species causing postharvest rot on Chinese cherry in China

Chinese cherry (Cerasus pseudocerasus) is an important native fruit crop with high economic and ornamental value. During April to July 2017 and 2018, Fusarium rot symptoms were observed on cold-stored Chinese cherry in China. The symptoms began as soft, water-soaked lesions, developed into soft with obviously sunken, decaying lesions covered by cotton-like white mycelia. Twenty-one fungal isolates were obtained from single spore cultures and identified as Fusarium spp. based on cultural and morphological characteristics. To confirm the identification, the internal transcribed spacer region (ITS) of ribosomal DNA and translation elongation factor 1-alpha (TEF-1α) sequences were amplified. Phylogenetic analysis showed that the ITS and TEF-1α sequences of the isolates clustered with available sequences at GenBank belonging to F.equiseti, F. fujikuroi, F. lateritium, F.proliferatum and F.acuminatum. Pathogenicity assays showed that all the Fusarium species could infect healthy fruits whether the fruits were wounded or not, cause the typical symptoms originally observed, and be re-isolated from inoculated fruits. Our study should be helpful for better understanding of Fusarium species causing postharvest rot on Chinese cherry in China, and will be useful for the prevention and control this disease.

First report of Fusarium oxysporum f.sp. cubense VCG 0125 and VCG 01220 of Race 1 infecting Cavendish bananas(Musa sp. AAA) in India.

Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is the most devastating disease affecting commercial and subsistence cultivation of banana (Musa spp.) worldwide. Generally, the Cavendish bananas are resistant to Foc race 1 that destroyed cv. 'Gros Michel' (AAA) and susceptible to tropical race 4 (TR4), which is causing severe epidemics in different banana-growing countries including India (Thangavelu et al. 2019). In 2019, a roving survey was conducted in major banana growing states of India such as Bihar, Uttar Pradesh, Gujarat and Tamil Nadu to assess the incidence of Fusarium wilt disease in Cavendish bananas and also to characterize the pathogens by different methods including Vegetative Compatibility Grouping (VCG) and molecular methods. The Fusarium wilt incidence in cv. Grand Naine (Cavendish group-AAA) was 6-65% in Bihar, 30-45% in Uttar Pradesh, 5-15% in Gujarat and 15- 21% in Tamil Nadu. For characterization, a total of 61 samples from the Fusarium wilt infected Cavendish bananas were collected and single spore culture of Foc was obtained. The morphological characterization revealed the presence of one to two oval- to kidney-shaped cells in false heads and sickle-shaped macroconidia and a foot-shaped basal cell. The pathogenicity was demonstrated by adopting randomized block design with five replications on cv. Grand Naine. The Koch's postulate was successfully completed by re-isolation of the inoculated Foc pathogen and characterization by PCR method. The VCG analysis carried out using nit-M testers of all known VCGs indicated the presence of VCG 0125 from the Foc samples collected from cv. Grand Naine grown in Uttar Pradesh (Siswabazar of Maharakanj district) and Tamil Nadu (Cumbum of Theni district), VCG 01220 from the Foc samples collected from cv. Grand Naine grown in Uttar Pradesh (Siswabazar of Maharakanj district) and Gujarat (Kamrej of Surat district,) and VCG 01213/16 from Foc samples collected from Uttar Pradesh (Siswabazar of Maharakanj district) and Bihar (Falka village of Katihar district) . The molecular confirmation of these VCGs 0125, and 01220 (Foc R1) isolates was carried out by PCR method using the primer set SIX6b_210_F and SIX6b_210_R (Carvalhais et al. 2019) for Foc R1, primer sets Foc TR4-F & Foc TR4 -R (Dita et al. 2010) for Foc TR4 and primer set Foc-1/Foc -2 (Lin et al. 2009) for Race 4. The results showed that only the primer set for Foc R1 has generated the expected amplicon size of 210 bp in the Foc isolates of VCG 0125 and 01220. Besides, the sequencing of Translation Elongation Factor (TEF) 1-α gene and BLAST searches in Genbank for the representative Foc isolates of VCG 0125 (Genbank no. MW 286800) showed 99.84% similarity to Foc R1 (KX365393.1) and Foc isolates of VCG 01220 (Genbank no. MW 286803) showed 99.69% similarity to Foc R1 (KX365413.1). Further, a phylogenetic analysis performed using the TEF1-α gene sequences showed that the Foc race 1 isolates (VCGs 0125 and 01220) from India were grouped with known Foc race 1 isolates from Tanzania and Australia. Based on the experimental results the study has confirmed the presence of VCGs 0125 and 01220 of Foc Race 1 in cv. Grand Naine in India. As these VCGs are most widely distributed and do not found to infect Cavendish bananas so far (Mostert et al. 2017), this report is very important from the quarantine and management perspectives. To the best of our knowledge, this is the first report of the occurrence of VCGs 0125 and 01220 of Foc Race 1 in cv. Grand Naine in India.

Micro propagation of Adiantum Capillus plant through a culture of plant spores

This experiment was conducted in Biotechnology research center BTRC for the purpose of studying the possibility of propagate Adiantum Capillus plant by using spores through plant tissues culture technology, which is one of the endangered plants in Libya. MS media was used in this study supplemented with some growth regulators (cytokines) of benzyl adenine BA and Kinten K at different concentrations (0.0, 0.5, 1.0, 2.0) mg / l. The results indicate that the growth of spores represented by the increase in the length and size of the vegetative masses in the MS nutritional medium equipped with BA growth regulator at a concentration of 2.0 mg / l is significantly higher than other treatments. The obtained plants were adapted by using growth medium of the Betmos and sand mixture in a ratio of 1: 2 in the growth room. The branches and roots formed when the plant moved to the greenhouse in the same medium of development

Molecular Identification of Trichoderma reesei.

Fungi comprise one of the most diverse groups of eukaryotes with many cryptic species that are difficult to identify. In this chapter, we detail a protocol for the molecular identification of the most industrially relevant species of Trichoderma-T. reesei. We first describe how a single spore culture should be isolated and used for the sequencing of the diagnostic fragment of the tef1 gene. Then, we provide two alternative methods that can be used for molecular identification and offerthe diagnostic oligonucleotide hallmark of the tef1 sequence that is present in sequences of all T. reesei strains known to date and that is therefore suitable for reliable and straightforward identification.

In vitro spore germination and early gametophyte development of Cibotium barometz (L.) J. Sm. in different media

Abstract. Isnaini Y, Praptosuwiryo TNg. 2020. In vitro spore germination and early gametophyte development of Cibotium barometz (L.) J. Sm. in different media. Biodiversitas 21: 5373-5381. Cibotium barometz (L.) J. Sm. is known as the golden chicken fern and included in Appendix II of CITES. It is an important export commodity for traditional and modern medicine. Globally, populations of this species are under significant pressure due to overexploitation in the wild. In vitro culture is one of the technologies used for ex-situ propagation and conservation of rare and endangered ferns and lycophytes. This study’s objectives were: (i) to observe in vitro spore germination and early gametophyte development of C. barometz, and (ii) to determine the best culture medium for rapid spore germination and early development of the gametophytes. The sterilized spores were sown in half-strength Murashige & Skoog (½MS) basal medium supplemented with combinations of 6-Benzylaminopurine (BAP) and α-Naphthalene acetic acid (NAA). A factorial combination of four BAP concentrations (0, 2, 4, and 6 mg L-1) with four concentrations of NAA (0; 0.01; 0.03 and 0.05 mg L-1) created 16 treatments replicated in a Completely Randomized Design. Spore germination of C. barometz was observed to be Vittaria-type, and its prothallial development was Drynaria-type. Spore germination started 7-14 days after sowing. Young heart-shape gametophytes consisting of 110-240 cells were formed in 45-61 days after sowing. The two best spore culture media for rapid spore germination and development of C. barometz gametophytes were ½ MS with or without 2 mg L-1 BAP.

Mycorrhizal inoculation impact on Acorus calamus L. - An ethnomedicinal plant of western Himalaya and its in silico studies for anti-inflammatory potential

Ethnopharmacological relevanceDifferent plants are used for the treatment of various ailments and Acorus calamus L. is one such plant found in Western Himalaya. Rhizome of this plants has ethnomedicinal significance, as its rhizome is used for curing fever, pain and inflammation. An attempt has been made to alter the phytochemicals and increase its antioxidant property in a sustainable way with the help of mycorrhizal inoculation. Aim of the studyStudy of mycorrhizal (Funneliformis mosseae) impact on the biological activities and phytochemical profile of A. calamus L. rhizome and in silico studies of phytochemicals for their anti-inflammatory property. Materials and methodsF. mosseae was mass multiplied by single spore culture and then A. calamus rhizomes were inoculated with it. Antioxidant potential of rhizome extract was observed by DPPH and FRAP assays and the phytochemical profiling was done with GC-MS analysis. For observing antimicrobial activity disc diffusion method was employed. Dominant phytochemicals α-asarone and monolinolein TMS were chosen for molecular docking studies against four receptors (4COX, 2AZ5, 5I1B, 1ALU). ResultsThere was increase in antioxidant activity of rhizome extract after mycorrhizal inoculation. However, no change in antimicrobial activity was observed in the plant after mycorrhizal inoculation. The comparison in phytochemicals was observed by GC-MS analysis which showed qualitative and quantitative variation in biochemical content in plants. The phytochemical, α-asarone and monolinolein TMS showed highest docking score and least binding energy against 1ALU and 4COX respectively for anti-inflammatory activity. ConclusionMedicinal plants are potential source of antioxidants which can be increased by mycorrhizal inoculation without addition of chemical fertilizers and also results in altering the phytochemical composition.

First Report of Colletotrichum salicis Causing Bitter Rot of Apple in Italy.

Apple (Malus domestica Borkh.) is the most important fruit crop in South Tyrol (northern Italy), with a total production of 905.089 tons in 2019 (Chamber of Commerce, Industry, Crafts and Agriculture of Bozen-Bolzano 2020). Symptoms of bitter rot were observed on organic apples of the cultivar 'Roho 3615'/Evelina® collected in a packinghouse in South Tyrol at the end of March 2018 after approximately six months of storage in controlled atmosphere. Lesions were circular and light brown with orange conidial masses. Tissue samples were removed under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to Petri dishes with potato dextrose agar (PDA), and incubated in the dark at room temperature for two weeks. Single spore cultures were obtained by adapting the procedure of Choi et al. (1999). Pure cultures were grown in quadruplicate on PDA at 20°C in the dark for two weeks. The colony appearance on the upper side was mostly flat with a distinct margin, the surface was covered with short, floccose aerial mycelium, and the color ranged from light gray to dark gray, while the reverse side appeared yellow. From each replicate culture, conidia were harvested, and the length and width of 50 randomly selected conidia were measured using a compound light microscope coupled to a digital camera (Leica DMLS, Leica Microsystems, Wetzlar, Germany). Conidia were cylindrical to fusiform, pointed at one end, and measured 10.0 to 19.5 × 2.5 to 5.0 μm (14.5 ± 1.9 × 3.9 ± 0.7 μm [mean ± SD]) in consistency with Damm et al. (2012). In order to determine the species of isolate 18-DSS-BS-EL-1-012, a multi-locus DNA sequence analysis was performed. Genomic DNA was extracted by following the protocol described by Cassago et al. (2002). Four loci, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3) and the internal transcribed spacer (ITS) region of the rRNA operon were amplified by PCR and Sanger sequenced (Damm et al. 2012; Lévesque and de Cock 2004). The obtained DNA sequences of ACT, GAPDH, HIS3 and ITS were 186, 150, 317 and 505 bp long and were submitted to GenBank under the accession numbers MT347599, MT347600, MT347598 and MT337388, respectively. A MegaBLAST analysis resulted in 100% sequence identity at all four loci with a type culture of Colletotrichum salicis (CBS 607.94; GenBank accession numbers: JQ949781, JQ948791, JQ949451 and JQ948460), which belongs to the C. acutatum species complex (Damm et al. 2012). A pathogenicity test was performed with twelve 'Golden Delicious' apples by wounding the fruit with a sterile piercing tool and inoculating 20 μl of spore suspension (104 conidia per ml) from a 21-day-old PDA culture. Inoculated fruits were incubated for 21 days in a moist chamber at 20°C in the dark. The symptoms were recorded at 3, 5, 7, 10, 14 and 21 days post-inoculation (dpi). Symptoms appeared after 7 days on all inoculated fruits and resembled those observed on the original fruit, while mock-inoculated controls with sterile water remained symptomless. Fungal colonies resembling the original culture were re-isolated from lesions on the apple and plated on PDA. Their identity was confirmed by DNA sequence analysis of the ITS region, thereby proving Koch's postulates. Bitter rot occurs globally and is considered one of the most important diseases of apple that has the potential to cause significant crop losses (Sutton et al. 2014). Other Colletotrichum species have been commonly reported from apple in Europe, such as C. godetiae and C. fioriniae, whereas C. salicis has been reported solely in New Zealand and Belgium (Damm et al. 2012; Grammen et al. 2019). To the best of our knowledge, this is the first report of C. salicis causing bitter rot of apple in Italy.

Fase gametofítica de tres taxones de Anemia (Anemiaceae)

Antecedentes y Objetivos: Anemiaceae presenta un único género, Anemia, con 115 especies que se distribuyen en el Neotrópico, África, India e islas en el Océano Índico. El objetivo de este trabajo es caracterizar las esporas vivas y la fase gametofítica de tres taxones de Anemia sudamericanas, poco estudiadas o con estudios fragmentados, A. herzogii, A. tomentosa var. anthriscifolia y A. tomentosa var. tomentosa.Métodos: Los gametofitos de los tres taxones han sido obtenidos a través de cultivos in vitro de las esporas. Las esporas y los gametofitos fueron estudiados a través del microscopio de luz y electrónico de barrido. Se describió el patrón de germinación de las esporas, evolución del desarrollo gametofítico y expresión sexual.Resultados clave: Los gametofitos filamentosos estaban formados por 4-20 células, los adultos fueron cordados simétricos o asimétricos, bisexuales y protándricos, en sus superficies y márgenes se observaron tricomas clavados. Los gametangios corresponden al tipo leptosporangiado. La dehiscencia de los anteridios se produjo mediante una perforación en la célula opercular. Los arquegonios tenían cuellos con cuatro a siete células y cuatro células triangulares en el extremo distal del cuello. Anemia herzogii mostró tricomas ramificados sobre los gametofitos y más tarde desarrolló esporofitos.Conclusiones: La germinación en todos los taxones corresponde al tipo Anemia y el desarrollo gametofítico al tipo Ceratopteris. Anemia herzogii se distingue de las dos variedades por la presencia de tricomas ramificados exclusivos de la fase gametofítica. Los taxones estudiados se caracterizan por la morfología del gametofito, tipo de tricomas y sexualidad.

Drynaria bonii spore culture: optimization of culture conditions and analysis of gametophyte and sporophyte development

Drynaria bonii H. Christ is a medicinal plant that has traditionally been used for the treatment of chronic diseases and tuberculosis. However, D. bonii has not yet been cultivated or propagated. In this study, we optimized the conditions for the spore culture of D. bonii to produce sporophytes. First, we evaluated the effects of various factors, such as temperature, light intensity, potting material, and pH, on spore germination and early gametophyte development. Under optimal culture conditions, 95–99% of the spores germinated within 2 weeks of culture. Next, based on these results, we established a culture system for gametophyte development and sporophyte production. Mature gametophytes first appeared 5 months post-germination (mpg), and at 12 mpg, the rate of production of heart-shaped gametophytes reached 56.6%. Sporophytes at the early first-leaf stage first appeared at 6 mpg, and at 12 mpg, the rate of formation of sporophytes reached 15.4%. Interestingly, 6.3% of all gametophytes produced multiple sporophytes. Additionally, 42–62% of the gametophytes also produced multiple secondary gametophytes, indicating a high potential of D. bonii gametophytes to generate new gametophytes and, subsequently, sporophytes. When transferred to new trays or pots, sporophytes grew well and showed 100% survival. Overall, we conclude that this spore culture system can be successfully used for the propagation of D. bonii sporophytes.

Reproductive potential and early spore settlement on different substrates in Gigartina skottsbergii (Gigartinaceae, Rhodophyta) from the South American Atlantic coast

The aims of this study were to estimate seasonal spore availability in the carrageenophyte Gigartina skottsbergii from the Patagonian Atlantic coast, evaluate the effect of induction methods and culture conditions on spore release, and assess spore settlement on different substrates. Fertile fronds were collected in southern Argentina. The density and size of reproductive structures and spore output were registered seasonally. Spore release was evaluated using different induction methods and conditions. Finally, different natural substrates were assessed for spore settlement. Spore availability is restricted to winter and spring. Cystocarps and tetrasporangial sori reached maturity towards winter, and spores were released in large amounts during winter and spring, with higher tetraspore than carpospore release. Spontaneous discharge would appear to be a more advisable induction method than desiccation and high salinity. Temperature and photoperiod did not have a significant effect on total spore output. After 2 weeks of incubation, no differences were found among spore densities on glass, pebbles, clams, or mussels. Despite the low survival rate registered on all substrates, spores germinated and developed healthily. This is the first study to evaluate carpospore and tetraspore availability throughout the year and the viability of in vitro spore culture of G. skottsbergii from the South American Atlantic coast.

Toxicological Studies and Histopathological Changes on Black Bean Aphid, Aphis craccivora Induced by Entomopathogenic Fungi, Metarhizium anisopliae and Purpureocillium lilacinum

Toxicological, histopathological and ultrastructure studies of Metarhizium anisopliae and Purpureocillium lilacinum infected Aphis craccivora nymph were investigated in this study. comparison between fungal spore suspensions and fungal culture filterates virulence effects of the two tested entomopathogenic fungi against A. craccivora nymph were done under laboratory condition and the toxicological bioassay results revealed that P.lilacinum has a high toxic effect than M. anisopliae in which the LC50 of fungal spore suspensions and culture filterates after 7 days post-treatment was (7×102, 1×105 spore/ml and 3.2, 4.7 % ) for P. lilacinum and M.anisopliae, respectively. Treated nymph with spore suspensions was examined using both light and transmission electron microscope to study histopathological and ultrastructure changes compared with the control one. This comparison suggested that there is a deformation of the nymph body, complete destruction of all internal organs with an extensive invasion of the fungal spores throughout all internal body regions.

Potential use of Claroideoglomus etunicatum to enrich signal grass (Brachiaria decumbens Stapf.) for silvopasture preparation

Silvopasture system improvement in managing post-mining land resources has been done by searching for a quality grass. One of the selected grass species is signal grass (Brachiaria decumbens Stapf.). This research aimed to prepare signal grass through the inoculation of AMF Claroideoglomus etunicatum, as an effort to enrich its growth before being applied to post-mining soil. Research stages included the AMF inoculation on signal grass through spore culture and then transferred the colonized grass to the pot using sterile zeolite as a growth medium. The treatment on the first stage was without and with AMF inoculation (dose of 20 spores) on signal grass which was repeated for 12 times. Incubation in a spore culture was 4 weeks while incubation in a pot containing sterile zeolite medium was 8 weeks. Research data were analyzed using the Shapiro-Wilk’s normality test, Independent Sample T-test, and Pearson’s correlation test. Observation results showed that the inoculation of C. etunicatum on signal grass was significantly impact on the increase of plant height, stem diameter, number of leaves, number of tillers, shoot and root fresh weight, and shoot dry weight (p <0.05). Microscopic observation showed that there was AMF colonization on treated signal grass roots in the amount of 55 ± 0.06 % with number of spores was 252 ± 9.82 per 10 g zeolites, while AMF infection was not found in uninoculated signal grass. It is expected that by providing signal grass inoculated with AMF C. etunicatum would support its growth in post-mining land for Silvopasture system.

Spore inactivation on solid surfaces by vaporized hydrogen peroxide-Influence of carrier material surface properties.

The aim of this study was to investigate the influence of surface hydrophobicity and roughness of carrier materials on the inactivation of bacterial spores with gaseous hydrogen peroxide whereas condensate formation is prevented. Spores of Bacillussubtilis and Bacillusatrophaeus were applied either as single spore culture or as a mixed spore population to simulate natural contamination with microorganisms of different characteristics. Inactivation with gaseous hydrogen peroxide was carried out at 5200ppm hydrogen peroxide without condensate formation. The inactivation results of B. subtilis and B. atrophaeus spores on carrier materials with varying surface hydrophobicity differed significantly. However, inactivation of the mixed spore populations resulted in similar resistance compared to the single spore batches. The results of this study indicate that surface hydrophobicity most probably has an impact on the inactivation with gaseous hydrogen peroxide whereas surface roughness only plays a minor role.

Роль дождевых червей в распространении сибирской язвы

The article discusses the issue of the danger of earthworms in the spread of anthrax in connection with the long-term persistence of the causative agent of the disease in the soil of cattle burial grounds. The results of assessing the effect of earthworms on soil cleansing from the anthrax pathogen and the properties of microorganisms isolated from worms are presented. Studies have shown that under the influence of worms, the amount of anthrax pathogen in the soil decreases by 30–50%, but the remaining spores did not change their properties and retained all the main biological and genetic factors of pathogenicity. Key words: earthworms, anthrax causative agent, spore culture, cultivation

Stable Tetrodotoxin Production by Bacillus sp. Strain 1839.

For the first time, tetrodotoxin (TTX) was detected in a bacterial strain after five years of cultivation in laboratory conditions since its isolation from the animal host. A reliable method suitable for bacterial samples, high-performance liquid chromatography with tandem mass spectrometry, was used for toxin detection in spore and vegetative cultures of Bacillus sp. 1839. TTX was detected in a spore culture of the strain.

Effects of the fungus Pestalotiopsis maculans (Ascomycota: Amphisphaeriales) on the gametophytic development of the fern Lygodium venustum (Lygodiaceae)

Lygodium sp. is a climbing fern that occurs in open forests, settling frequently in areas with disturbed vegetation. In Argentina, Lygodium venustum inhabits in the protected area "Moconá" Provincial Park, in few and small populations. In-vitro cultivation of spores is a strategy for ex-situ conservation of species with reduced or threatened populations. Usually, fern spores need to be surface sterilized before sowing, to avoid the development of unwanted microorganisms. As part of our project focused on the conservation of native fern species in Argentina, the aim of this work was to evaluate a propagation protocol for L. venustum through in-vitro cultivation of spores, employing a standard procedure of disinfection, to investigate its effect on spore germination and the subsequent gametophyte as well as its response by the inoculation with a fungus. A batch of spores was previously treated with 10% (v/v) solution of NaOCl during 2 min and then sown in Dyer agar medium supplemented with 1% Nystatin (T1). The petri dishes were incubated in a growth chamber under white illumination (28μmol m-2 s-1) with a photoperiod of 12 hr and a temperature of 22±2°C. Another set of spores without previous disinfection was sown on Dyer agar medium and under the same conditions and used as a control (T0). Spore germination and gametophyte and sporophyte development were monitored through periodical observations under stereoscopic and light microscopes. As during cultures of non-disinfected spores a fungus developed in association with L. venustum gametophytes, its conidia were morphologically characterized as well as those obtained from the black spots present on the sporophyte leaves. A conidial suspension was applied to 20 days-old gametophytes obtained in axenic conditions to evaluate the appearance of any symptom. In both treatments (T1 and T0), spores germinated at 7 days after sowing. The pattern of spore germination followed the Anemia-type, unlike the Vittaria-type found by other researchers. Untreated spores showed a higher mean percentage of germination than those treated (86 and 72 % in 30 days, respectively). In T0 gametophytes grew in association with a fungus, whose conidia had the morphological characteristics of Pestalotiopsis maculans, as occurred with the conidia obtained from the black spots of L. venustum leaves. The association of P. maculans with L. venustum is reported here for the first time. The gametophytes developed in T0 and those inoculated with P. maculans became necrotic and died before producing gametangia, suggesting a pathogenic role of the fungus. The elevated percentage of spore germination registered in T0 could also indicate some effect of P. maculans or its metabolites in the promotion of spore germination. Gametophytes developed in T1 became bisexual or unisexual male, which points to the presence of some antheridiogen that induces the production of antheridia on smaller or slower-growing gametophytes. The sporophytes arose after 4 months of spore sown in T1, pointing out the methodology employed here suitable for the ex-situ conservation of L. venustum.

PENINGKATAN EFISIENSI PENGGUNAAN LAHAN KELOMPOK TANI SUNGKAI PERMAI MELALUI TUMPANG SARI JAGUNG DAN KACANG TANAH DAN PENGENALAN CENDAWAN MIKORIZA ARBUSKULA (CMA)

The Sungkai Permai Lambung Bukik Farmer Group, Padang City, generally cultivates in monoculture, with limited productive land. The land is planted with rice, secondary crops such as corn, and some peanuts. In the past two years, corn farmers often experience crop failures, especially during the rainy season, where the sun shines less due to global climate change. Therefore, diversifying commodities on limited land so that land efficiency increases are significant to prepare the next source of seeds. To support increased land use efficiency, optimal soil conditions are needed. Utilization of Arbuscular Mycorrhizal fungi can improve soil fertility and is expected to become an alternative technology in plant cultivation. Science and technology activities based on the Study Program and the Assisted Nagari (IbPSNB) were carried out in groups of Permai Sungkai Farmers in the form of 1) Counseling on the pattern of corn-peanut intercropping cultivation, and how to make CMA spore cultures, 2). The demonstration plot for corn-peanut intercropping cultivation. Through this activity, farmers finally understood the benefits and patterns of corn-peanut intercropping. Farmers understand the role and method of making CMA spore cultures. Several farmers cultivated corn-peanut intercropping. Two farmers at the demonstration plot managed to harvest corn and peanuts at the same time. Other farmers have failed to harvest corn due to pest attacks but have succeeded in harvesting peanuts so that the losses experienced by farmers are reduced, and they still have stock of seeds for the next planting season.

Biological control of Paecilomyces formosus, the causal agent of dieback and canker diseases of pistachio by two strains of Streptomyces misionensis

Dieback and canker of pistachio shoots and limbs is a commercially significant disease in Iran. Paecilomyces formosus (Sakag., May., Inoue and Tada) Houbraken & Samson is considered as the major causal agent of this malady. Actinomycetes and particularly members of the genus Streptomyces are well known as biocontrol agents for many plant diseases; they achieve this by production of bioactive metabolites and hydrolytic enzymes. The present study aimed to exploit Streptomyces isolates to biologically control P. formosus in vitro and in greenhouse experiments. Fifty two actinomycetes isolates were evaluated for their antagonistic effect against P. formosus isolated from diseased orchards. Considering their in vitro antagonistic performance, two isolates, designated as BH4-1 and BH4-3 were selected for molecular identification and in vivo experiment to be evaluated for biocontrol efficacy. Partial sequences of 16S rDNA unveiled their identity as two strains of Streptomyces misionensis with 99.9% sequence homology. For in vivo greenhouse experiments, inoculation of artificially made wounds on pistachio young trees stems performed with spore suspensions and culture extracts of either of the two bioactive S. misionensis Cercos et al. strains alone and/or together with P. formosus spore suspensions. Cupravit® was used as fungicide control. To evaluate the effect of the biocontrol test agents on the development of disease symptoms and severity, after about nine months, canker severity score, length and width of internal discolored lesions and frequency of P. formosus re-isolation from lesion margins were measured. The recorded disease criteria for treatments receiving S. misionensis strains were significantly in favor of bio-controlling the disease compared to the controls.

Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating.

The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of B. subtilis form sub-populations which include vegetative cells, sporulating cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Automated gating procedures using Gaussian mixture modeling (GMM) were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in germination deficient strains harboring different genome modifications. A decrease in the sporulation efficiency of strain Bs02018, utilized for the display of sfGFP on the spores surface was observed. On the contrary, a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA (Bs02025) exhibited the highest sporulation efficiency, as within 24 h of cultivation in sporulation medium, cultures of BS02025 already consisted of 80% spores as opposed to 18% for the control strain. We confirmed the identity of the different subpopulations formed during sporulation by employing sorting and microscopy.