Wet Cotton Wool Research Articles

First report of Lasiodiplodia theobromae causing dieback in custard apple (Annona squamosa) tree in China.

Custard apple (Annona squamosa Linn) is popular for its sweet taste and rich aroma. Hainan province is the major production area of custard apple in China. In September 2020, wilting of leaves and branches, discoloration of the vascular system and dieback of trees were observed in plantings in Lingao County of Hainnan Province, China (Fig. 1a-c). The incidence of dieback in three orchards was at least 19%, and affected samples were brought to the laboratory. Fragments of approximately 5 mm in length were obtained from five diseased branches, which were collected from different plants and orchards. Fragment surface were sterilized in 75% ethanol for 1 min, and 1% mercury chloride for 1 min, then rinsed three times with sterile distilled water. These tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated at 28°C for 3 days. Fungal colonies were transferred to fresh PDA plates, and single-spore cultures were obtained. We isolated twenty-six fungal strains, of which twenty-three isolates were morphologically identified as Lasiodiplodia species (Phillips et al. 2013). The colony morphology was initially round and white, then turned to grey or black after 5-7 days at 28°C, and formed pycnidia for 20 days (Fig. 1d). The immature conidia were ellipsoid, colorless, hyaline and unicellular, becoming brown, bicellular with longitudinal striations at maturity (Fig. 1e). Mature conidial size was: 26.61±1.57×14.87±1.14 µm (n=60). For molecular identification, genomic DNA of three isolates (HSYF01, HSYF02 and HSYF03) was extracted using E.Z.N.A.® HP Plant DNA kit (Omega Bio-Tek). The internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α) and β-tubulin (TUB) regions were amplified using the primers ITS1/4, EF1-728F/986R and Bt2a/Bt2b, respectively (Carbone and Kohn 1999; Glass and Donaldson 1995; White et al. 1990). A BLAST search of ITS, EF1-α and TUB gene sequences (Accession nos. MW625913-MW625918, MW876481-MW876483) had in 99.8% (493bp out of 494 bp), 99.31% (286bp out of 288 bp) and 100% (372bp out of 372 bp) identity to CBS164.96 of the L. theobromae (Accession nos. AY640255, AY640258 and KU887532). The identity of the putative pathogen isolates was further confirmed by phylogenetic analysis (Fig. 2). Ten healthy 1-2-year-old custard apple trees were used to confirm pathogenicity. Custard apple plants were wounded approximately 15-20 cm below the tips with a sterilized scalpel. Each cut was inoculated with a 5 mm agar plugs with mycelium from the PDA cultures. The wound site was moisturized with wet cotton wool and wrapped with laboratory film (Silveira et al. 2018). Two seedlings treated with sterile agar plugs served as a controls. The pathogenicity test was repeated twice. After 14 days of incubation in a glasshouse, all inoculated seedlings had characteristic discoloration and necrotic lesions starting from the apical branches, (Figs. 1f and 1g). The stems exhibited browning and vascular streaking of the wood from the inoculation point (Figs. 1h), while the control seedlings remained symptomless. Typical colonies of L. theobromae were isolated and identified from all inoculated seedings, fulfilling Koch´s postulates. Although, postharvest fruit stem-end rot on custard apple caused by L. theobromae was previously described (Hu et al. 2003; Meng et al. 2017), this is the first report of L. theobromae causing dieback in mature custard apple trees in China.

First report of Phaeoacremonium oleae and P. viticola associated with olive trunk diseases in Italy.

Over 300 trunk, branch and stem samples with vascular discolouration, necrotic wood and shoot death were collected from olive (Olea europaea) orchards in Lecce, Brindisi, Bari and Foggia provinces (Apulia region, Italy) from October to May from 2013 to 2019. Small chips of symptomatic wood samples were surface sterilised (5% NaOCl, 3 min; 70% ethanol, 30 s), rinsed (sterile distilled water, ×3), and placed onto potato dextrose agar (PDA) plates amended with 500 ppm streptomycin sulphate. After 14 days at 25 °C in the dark, hyphal tips of growing fungi, including different taxa, for instance Phaeoacremonium and Botryosphaeriaceae spp., were transferred to new PDA plates and incubated until sporulation. Monoclonal colonies resembling Phaeoacremonium-like genus (Mostert et al. 2006) were selected for further study, and genomic DNA of 59 representative isolates was extracted (Carlucci et al. 2013). Partial actin and β-tubulin genes were amplified with primers ACT-513F/ACT-783R (Carbone & Kohn 1999), and T1(O'Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson 1995), respectively. The sequenced amplicons were compared by BLAST algorithms with reference strains of Phaeoacremonium spp. retrieved from GenBank. Forty-four isolates showed 99% to 100% similarity with reference strains P. italicum, P. minimum, P. parasiticum, P. scolyti and P. sicilianum (Carlucci et al. 2015), nine with P. oleae, and six with P. viticola. Actin and β-tubulin sequences of P. oleae (Pm14) and P. viticola (Pm34) were submitted to GenBank (MW714561, MW714563; MZ318697, MZ318696). Microscopy of P. oleae isolates showed: conidiophores branched and unbranched, (18.7-)21.9-57.1(-67.8) × (2.9-)3.3-4.7(-5.2) (mean, 38.9×4.1) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.4 to 5.5 μm long, and 1.5 to 2.4 (mean, 4.6 × 2.2) μm wide (n=30). Microscopy of P. viticola isolates showed: conidiophores subcylindrical, branched at base (6.7-)8.9-27.2(-29.3) × (2.0-)2.6-3.3(-3.7) (mean, 21.4 × 3.2) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.3 to 6.8 μm long, and 1.1 to 2.2 (mean, 4.2 × 1.6) μm wide (n=30). In spring 2020, artificial inoculations were carried out with P. oleae (Pm14, Pm46) and P. viticola (Pm34, Pm43) strains on 10 healthy, 2-year-old olive seedlings cultivar 'Coratina'. Agar plugs (diameter, 0.3-0.5 cm) from 10-day-old cultures grown on water agar at 23 (±2) °C were inserted under the bark of small wounds in the stems (length, 0.4-1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten control olive seedlings were inoculated with sterile agar plugs. The experiment was replicated three times. All inoculated young olive plants were grown in pots in a greenhouse without temperature control. After 120 days, inoculated plants showed decline symptoms, and when cut longitudinally, brown streaks were observed in the wood. For P. oleae these streaks measured 3.0-5.5 cm long (standard deviation [SD], 0.9 cm, and for P. viticola they were 1.8-3.5 cm (SD, 0.62). Both fungal species were re-isolated from the symptomatic wood from 85% and 80%, respectively, of these inoculated olive seedlings, fulfilling Koch's postulates. No symptoms were observed from olive seedlings used as control. P. oleae was first described as a fungal pathogen of wild olive (Olea europaea subsp. cuspidate) in South Africa by Spies et al. (2018), and P. viticola as a fungal pathogen of grapevine in France by Dupont et al. (2000). To the best of our knowledge, this is the first report of P. oleae associated with olive trunk disease in Italy, and the first report of P. viticola associated with olive trunk disease worldwide.

First report of Phaeoacremonium amygdalinum associated with almond dieback and wood disease in Italy.

In the last 10 years, almond (Prunus dulcis (Mill.) Webb) cultivation areas in the region of Apulia (southern Italy) have increased. A recent survey in young almond orchards recorded characteristic disease symptoms including rapid collapse of branches, chlorosis of leaves (with sudden wilting and death) in June/July, and bud and shoot dieback in December-March. Internal wood symptoms on stems consisted of brown vascular streaking that led to death of young apical shoots within a few weeks. Wood samples were collected and taken to the laboratory, where they underwent disinfection according to Fisher et al. (2002). Fungal isolation techniques were applied, with small pieces of wood tissue (0.5-1.0 cm) placed on potato dextrose agar (PDA; Oxoid Ltd.) supplemented with 300 mg/L streptomycin sulphate, and incubated at 23 ±2 °C in the dark. After 10 days, based on morphological features carried out by microscopic studies, 85% (n = 55) of the resulting fungal colonies (N = 65) were preliminarily attributed to known fungal pathogens of woody tissues, of which 12.4% (n = 8) belonged to the Phaeoacremonium genus. These last were purified by transferring single germinating conidia onto fresh PDA plates. In particular, the conidiophores were mostly short, usually unbranched, erect to flexuous, up to 5-septate, brown to pale brown, verruculose on the lower part, (12-)15.7-41(-55) (av. -29) μm long and 1.6-3.2 (av. 2.1) μm wide (n = 30). Conidia hyaline, oblong ellipsoidal-obovoid or subcylindrical (3-)4-5 μm long and 1.5-3 (av. = 4.4 × 2.1) μm wide (n =30). All Phaeoacremonium isolates were successfully subjected to genomic DNA extraction, according to Carlucci et al. (2013). Partial actin and β-tubulin genes were amplified with the ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) primers, respectively. Consensus sequences were compared with reference strains (extype) in the GenBank database, using the Basic Local Alignment Search Tool (BLAST). In particular, all isolates showed 99-100% similarity with reference strains of Phaeoacremonium amygdalinum Gramaje, Armengol & L. Mostert (JN191301; JN191305). Actin and β-tubulin sequences of P. amygdalinum strain Pm10 were deposited in GenBank (MW716265; MW714562). In spring 2020, artificial inoculations with two isolates of P. amygdalinum (Pm10; Pm15) were carried out on 10 healthy, 2-year-old almond seedlings of cv. 'Filippo Cea'. Agar plugs (diameter, 0.3-0.5 cm) taken from 10-day-old cultures grown on PDA at 23 ±2 °C were inserted into small wounds under the bark of the stems (length, 0.4-1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten almond seedling were used as controls, being inoculated with sterile agar plugs. The experiment was replicated three times. The inoculated young almond plants were grown in pots in a greenhouse without temperature control. After 150 days, the inoculated plants showed decline symptoms and internal longitudinal brown wood streaking (average, length, 2.7-6.4 cm). Phaeoacremonium amygdalinum was reisolated from symptomatic wood of 95% of the experimental almond seedlings, thus fulfilling Koch's postulates. No symptoms were observed on almond seedling used as controls. Phaeoacremonium amygdalinum was first described as a fungal pathogen of almond in Spain (Gramaje et al., 2012). To the best of our knowledge, this is the first report of P. amygdalinum associated with almond dieback disease in Italy. References Carbone I. and Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Fisher et al 2002. Mycol. Prog. 1:315 Glass N. L. and Donaldson G. C. 1995. J. Cl. Microbiol. 41:1332. Gramaje et al. 2012. Persoonia 28:1. O'Donnell K. and Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103.

Pulpal dressing condensation methods in pulpotomy for primary molars: An in-vitro comparative study

PurposeTo compare three condensation techniques of zinc oxide eugenol (ZOE) as a pulpal dressing material during pulpotomy in extracted primary molars. Material and MethodsSixty primary first and second molars were embedded in individual wax casts and divided into three groups consisting of 20 teeth each. In group I, the ZOE base was condensed by an amalgam condenser, while a moist cotton pellet was used in group II. A combination of an amalgam condenser and a wet cotton pellet tested the condensation mentod in group III. The condensation quality of the three techniques was evaluated through two digital periapical radiographs taken in a lateral and anterior-posterior direction. ResultsNon-parametric Kruskal-Wallis test showed that there was no significant difference between the technique and quality of ZOE compaction. However, a significant difference was observed on condensation assessment for combined three groups on lateral radiographs vs the appearance on antero-posterior radiographs with the p-value set at < 0.05. ConclusionVoids appeared with all three techniques. A combination of an amalgam condenser and the wet cotton wool pellet was the least effective method of condensation. Lateral radiographs revealed much fewer spaces between the ZOE and pulpal floor in comparison to antero-posterior images.

Effects of light-emitting diodes (LEDs) on lipid production of the aerial microalga Coccomyxa sp. KGU-D001 under liquid- and aerial-phase conditions

Algal biofuels are a promising alternative to fossil fuels, but their widespread use is hindered by problems with mass production. Light-emitting diodes (LEDs) with specific light wavelengths could be used as an energy source for algal growth and lipid synthesis. In this study, the effects of light source on the biomass and lipid production of the aerial microalga Coccomyxa sp. KGU-D001 were evaluated using LEDs. The integration of two-phase cultures, including growth and lipid production under the stress of nitrate depletion, was assessed for efficient lipid production under liquid- or aerial-phase conditions. Different wavelengths of light (blue, green, and red) were tested under liquid- and aerial-phase conditions. Under aerial-phase culture, the fatty acid contents in biofilm reached 320 mg g DWC−1 with the red LEDs. In view of these findings, we describe a one-step culture method for growth and lipid accumulation in algal biofilm under aerial-phase culture with red LED irradiation. When Coccomyxa biofilm was cultured on wet cotton wool with BBM in a petri dish under the red LED, it was able to grow and accumulate lipids under the aerial-phase condition. Based on the results of this study, a potential method for a continuous biodiesel production system is proposed.

First report of Lasiodiplodia theobromae causing decline of blueberry (Vaccinium corymbosum L.) in the Czech Republic.

Popularity of blueberries (Vaccinium corymbosum L.) has been rising worldwide, due to their nutritional bioactive compounds such as flavonoids, anthocyanins and polyphenols (Sinelli et al. 2008). These factors led to an increase of the demand for fresh blueberries in the Czech market, which resulted in increased blueberry planted in the Czech Republic. In spring 2018, about 200,000 1-year-old blueberry plants originally from the Netherlands were planted on 47 ha in the South Moravian region. In September 2019, wilting of leaves and shoots, bark necrosis and dieback of the plant were observed. The occurrence of blueberry decline symptoms in the field was near 2% of 5,000 observed plants which resulted in significant yield losses at the beginning of the production. Twenty shoots of symptomatic plants were collected (one shoot from each plant, twenty plants in total) and surface-disinfected using 75% ethanol. Necrotic tissues of xylem were aseptically placed on potato dextrose agar (PDA) (Himedia, India) amended with streptomycin and incubated at 25°C in the dark for one week. Nine isolates of Botryosphaeriaceae-like fungi were obtained from twenty plants and were transferred on PDA. Aerial mycelium grew rapidly, initially white, and then became grey to black. To enhance the conidia production, the isolates were transferred on water agar (Himedia, India) with autoclaved poplar twigs, placed near day light and incubated at room temperature. Three weeks later, black pycnidia were observed on the poplar twigs. Mature pycnidia exuded white/grey coloured masses of conidiospores. Conidia were granular, subcylindrical to elliptical. Immature conidia were single-celled and hyaline measuring 9.73 ± 1.39 × 16.86 ± 1.81 µm (n=60). Mature conidia were thick-walled with a septum from 11.30 ± 1.17 × 16 ± 1.58 µm (n=60). Based on conidiogenous cells, paraphyses and longitudinal striations on mature conidia characteristics, the isolates were morphologically assigned to the Lasiodiplodia genus (Phillips et al. 2013). Isolates were single-spored and total DNA was extracted with NucleoSpin Tissue (Macherey-Nagel, Germany) following manufacturer's protocol. ITS region, including the 5.8S, and part of the translation elongation factor 1 alpha (TEF-1α) were amplified with ITS1/ITS4 and EF1-728F/EF1-986R primers, respectively (Carbone and Kohn 1999; White et al. 1990). Consensus sequences were blasted using NCBI nucleotide BLASTn search. The sequences of ITS region were in 468/469 nts (MEND-F-00167) and 486/490 nts (MEND-F-00168) similar to Lasiodiplodia theobromae isolate CBS 287.47 and 286/289 nts (MEND-F-00167), 287/290 nts (MEND-F-00168) in case of TEF-1α. Sequences of two representative isolates (MEND-F-00167 and MEND-F-00168) were deposited in GenBank under accession numbers MN983133 and MN983134 for ITS and MN989914 and MN989915 for TEF-1α. For pathogenicity tests, 6 mm agar plugs with mycelium obtained from ten day old mycelia of both fungal isolates were inoculated on ten 1-year-old blueberry shoots cv. Top Shelf collected from one plant, using a cork borer. Five shoots and pure agar plugs were used as a control. Plugs were covered with wet cotton wool to keep moisture and fixed with Parafilm. After three weeks, characteristic discoloration and lesions appeared on the inoculated shoots. Control shoots remained symptomless. Necrotic tissue was aseptically cut and L. theobromae was re-isolated on PDA. Identity of fungus was confirmed by morphological characterization and sequencing, fulfilling Koch´s postulates. Blueberry decline caused by Botryosphaeriaceae spp. has already been described around the world (Hilário et al. 2020; Tennakoon et al. 2018; Wiseman et al. 2017; Xu et al. 2015). To our knowledge, this is the first report of L. theobromae on blueberry in the Czech Republic.

First Report of Cladosporium tenuissimum Associated with Leaf Spot of Alfalfa (Medicago sativa) in China

Alfalfa (Medicago sativa L.) is a leading forage crop that is known as the “king of forage.” It has been widely cultivated all over the world for its high nutritional quality and high biomass production (Liu et al. 2018). Since 2015, an unknown leaf spot symptom with approximately 18% disease incidence has been observed on M. sativa at a pratacultural science experimental field of Southwest University, 105.58°E, 29.40°N, Chongqing, China. Infected leaves gradually exhibited white to gray irregular necrotic lesions. In severe cases, some leaf spots coalesced, covering more than 50% of the leaf area, eventually causing the leaves to fall off. The leaves with the spot symptoms were collected in field, surface sterilized with 70% ethanol for 5 s followed by 0.1% HgCl₂ treatment for 4 min, and rinsed in sterile water three times. Thereafter, leaf samples (2 × 2 mm) from margins of individual lesions were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. The diameter of the fungal colonies was 4.2 cm (3.4 to 5.3 cm) (n = 6). From a top-down view, colonies on PDA usually exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Colony color ranged from olive-brown to dark green; margins varied from gray-olivaceous to white without prominent exudates, and sporulation was profuse. From a bottom-up view, the colony color appeared almost dark. Hyphae were branched, 2 to 5 μm wide, pale olivaceous-brown, often irregular in outline, sometimes constricted at the septa. Conidiophores were subcylindrical-filiform, unbranched, 320 μm (46 to 510 μm) × 3.4 μm (2.5 to 4.1 μm) (n = 30), tips often unilaterally swollen. Ramoconidia were zero to one septate, smooth walled, 17.5 μm (9.3 to 24.6 μm) × 3.8 μm (2.8 to 5.0 μm) (n = 30). Conidia were single celled, smooth, olive-brown, and elliptical to limonifor, 4.5 μm (2.8 to 6.1 μm) × 3.0 μm (2.2 to 3.7 μm) (n = 100). Based on morphological and growth characteristics, the features of associated fungal pathogens were consistent with those of Cladosporium tenuissimum (Bensch et al. 2010, 2012). To further confirm its identity, three different genomic DNA regions—the internal transcribed spacer (ITS) regions, partial translation elongation factor-1 alpha (EF), and actin (ACT)—were amplified and sequenced. Fungal genomic DNA was extracted, and the ITS, EF, and ACT were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R (Bensch et al. 2012; Jo et al. 2018). The ITS sequence showed 99.6% identity with that of C. tenuissimum (MH383051.1). DNA sequences from EF and ACT were 100% identical to those of C. tenuissimum (MF473728.1 and LN834588.1). The representative sequences were deposited in GenBank (accession nos. MK311328, MK341044, and MK341045). Based on morphology and DNA sequence analysis, the associated fungus was demonstrated to be C. tenuissimum. Pathogenicity trials were conducted in a greenhouse at 25 ± 3°C under natural daylight conditions. Fungal isolates were grown on PDA for 7 days. Spores were harvested and resuspended in sterile water (2.0 × 10⁶ conidia/ml). Six pots of M. sativa (cv. Eureka) were inoculated by spraying on the leaves with conidial suspension. Another six control pots were sprayed with sterile water. All pots were covered with plastic bags containing wet cotton wool for 48 h. Seven days after inoculation, the inoculated plant leaves showed leaf spot symptoms that were similar to those previously observed in the field. From the leaf spots of inoculated plants, C. tenuissimum was successfully isolated again. Control plants did not exhibit any disease symptoms. To our best knowledge, this is the first report on the occurrence of C. tenuissimum causing leaf spot on M. sativa in China. So far, this pathogen has not been found to be able to cause plant death. However, it can damage the leaves of alfalfa and affect forage quality and yield. Understanding its etiology may help to control this plant fungal pathogen, thus reducing economic losses.

THE SURFACE OF THE LEAF BLADE AS A FACTOR INFLUENCING THE SPIDER MITES DEVELOPMENT ON CUCUMBER

A comparative assessment of spider mite eggs development on the upper and under side of cucumber cotyledons has been made under laboratory experiments on the varieties Ginga F1 and Vyaznikovsky 37. Studies have been conducted on the cotyledons cut off from growing plants and placed on a wet cotton wool in the Petri dish. Mite eggs laid by females for five hours have been used for work. It has been shown that mites develop more slowly on the underside of cotyledons in comparison to the upper side when cotyledons are not exposed to spider mite. This may be due to the peculiarities of gas exchange on the abaxial and adaxial sides of leaves. It has been also found, that the mite embryonic development duration is increased, if occurs on the underside of the cotyledon , damaged by the spider mite within 1 day. Apparently, this is caused by the plant defense reactions to herbivory occurring increasingly on the lower side of cotyledons and associated with volatiles that has adverse effect on the mite eggs development. It is assumed that responses to the spider mites damage in the cucumber cotyledon leaves (in particular, those associated with the releasing of volatile compounds) are more characteristic for the spongy parenchyma cells than for the palisade cells in the leaf mesophyll.

CO2 fixation and lipid accumulation in biofilms of the aerial microalga Coccomyxa sp. KGU-D001 (Trebouxiophyceae)

This study compares lipid accumulation by immobilized aerial microalga Coccomyxa sp. KGU-D001 (Trebouxiophyceae) in biofilms and in aqueous suspended cultivation. Under direct exposure to normal air (low CO2) and light conditions, triacylglycerol (TAG) synthesis was significantly stimulated in algal biofilms on wet cotton wool. The TAG accumulation increased with decreasing thickness of the biofilm. This trend was attributed to CO2 fixation by photosynthesis of the biofilm cells, which increases the cellular accumulation of TAG. In a 17-μm-thick biofilm cultivated for 7 days, the total fatty acid (FA) content reached 27% of the cell dry weight. Under these conditions, approximately 60% of the fixed cellular carbon was presumed to be invested in TAG synthesis through free FAs. The CO2 fixation rate was almost maximized at relatively low photon flux density (25 μmol photons m−2 s−1). CO2-gas fixing algal biofilm systems can realize novel non-suspended cultivations, called artificial leaves, and thus present as strong candidates for biodiesel production in the near future.

Low Cost Quantitative Arsenic Determination Kit: A Method by Using Silver Nitrate

Abstract Arsenic sometimes found in ground water which has adverse effect on human health at levels as low as 10µg/ L. Arsenic is known to be one of the most toxic element that cannot be destroyed once it has entered to the environment. It is a dangerous poison at high doses but can cure diseases such as syphilis and leukaemia at low doses. The present study comprises a low cost technique for determination of arsenic concentration in groundwater up to 1000µg/L. The method is based on the reaction of arsenic (V) with potassium iodide and stannous chloride in concentrated hydrochloric acid medium to convert to arsenic (III) and needs 15 to 20 minutes time for complete conversion. After reaction with the granulated zinc (arsenic free), the evolved arsine gas is passed through wet cotton wool of lead acetate for absorbing any sulphide impurities and is finally absorbed by wet filter paper of silver nitrate which gives brown colour. The intensity of the colour varies with respect to the arsenic concentration present in ground water. A master colour chart is prepared by the known arsenic standard solution from 0 to 1000 µg/L for measuring the concentration range of arsenic in contaminated ground water. The quantitative arsenic determination is done with the help of colorimetric study. The absorbed colour of filter paper is thoroughly scanned and Red, Green and Blue (R-G-B) values are obtained by colour picking technique with the help of the software ‘Microsoft Paint’ under the operating system ‘Microsoft Windows 7’. The known arsenic concentrations are plotted against the R-G-B values of the corresponding absorbed colour and three master curves are drawn by the software ‘Mat lab’ for measuring the exact concentration of arsenic in groundwater. For validation, arsenic contamination test is conducted for the ground water samples collected from different areas of 24 Parganas district using our very low cost kit and other costly instruments like Digital Arsenator and Atomic Absorption Spectrometer.

First Report of Cane Blight on Blackberry Caused by Diaporthe eres in Croatia.

A cane disease of blackberry (Rubus sp.) cv. Thornfree was observed in May and June 2010 in two growing regions in the eastern part of Slavonia in Croatia. Symptoms consisted of bleached areas between and around cane nodes with some canes showing wilt symptoms. Infected areas were covered with numerous, black pycnidia immersed in the epidermal tissue. Disease occurrence in orchards growing cv. Thornfree ranged between 1 and 15%. Thirty disease samples were collected, disinfected (1 min in 70% ethanol and 2 min in 1% NaOCl), and placed in a moist chamber for 4 days. Fungal sporulating structures were then picked off and placed on potato dextrose agar (PDA). Fungal isolates obtained were identified as a Phomopsis sp., the conidial state of Diaporthe (3), on the basis of cultural and morphological characteristics. Alpha conidia were unicellular, hyaline, fusiform, sometimes tapering toward one or both ends, biguttulate (sometimes with several guttules), and 5.2 to 9.7 × 1.4 to 2.7 μm (average 6.5 × 2.1 μm). Beta conidia were hyaline, aseptate, filiform, hamate, and 16.6 to 28.2 × 0.5 to 1.5 μm (average 24.0 × 1.1 μm). The teleomorph was not observed. Biomolecular analysis was performed to identify the fungal species by sequencing the internal transcribed spacer (ITS) region spanning ITS 1, 5.8S rDNA, and ITS 2 of two isolates (Phk1 and Phk2). The amplified product was sequenced (GenLab-Enea, Rome, Italy) and a BLAST search of the NCBI nucleotide database was performed. Sequences from Phk1 and Phk2 (GenBank Accession Nos. HQ533144 and HQ533143, respectively) were identical to authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514, BPI 748435, and CBS 109767) ITS sequences in GenBank. Fungal isolates for pathogenicity tests were grown on PDA at 25°C for 7 days (12 h light/dark regimen). Inoculations were made on 30 to 40 cm long green shoots of potted plants of the blackberry cv. Thornfree. One-centimeter long wounds were made with a sterile scalpel and mycelia of D. eres were placed in the wounds. Inoculation sites were covered with a piece of wet cotton wool and aluminum foil to retain moisture. Three replications of 10 plants each were inoculated and these plus 10 control plants (inoculated with plugs of PDA only) were maintained in a growth chamber at 25°C. After 25 days, lesions developed on all 30 inoculated plants that averaged 15 mm long and control plants remained symptomless. D. eres was reisolated from inoculated plants, thus completing Koch's postulates. Phomopsis spp. have previously been reported on blackberry canes in Serbia (1) and Yugoslavia (2,4), however, to our knowledge, this is the first report of the occurrence of D. eres (anamorph P. oblonga) on blackberry in Croatia.

Water-independent effects of dietary restriction in Drosophila

Dietary restriction (DR) is a moderate reduction in food intake, without malnutrition, that extends the healthy life span in many organisms, including Drosophila (1). Although fruit flies have many practical advantages for studying DR, various technical complexities can have large effects on experimental outcomes (1). For DR, it is important that the basic food conditions are optimal. This ensures that increased longevity due to food restriction prolongs a healthy life span rather than returning sick animals to normal health by limiting access to a nutritionally inappropriate diet. We have systematically optimized our conditions for Drosophila DR, eliminated several non-nutritional explanations including water imbalance, and recommend a Brewer's-yeast-based diet (2, 3). Ja et al. (4) report contradictory data. Here, we present additional data in support of our conclusions and point out a flaw in the data concerning the Brewer's yeast diet presented by Ja et al. First, we developed a system that effectively hydrates flies under salt stress (Table 1, experiment 1). Adding 8 g·L−1 NaCl to our standard food [1.0 SYBrewer's (2)] shortened median life span by 24%. Adding to each vial a 200-μl pipette tip filled with water (1% agar) restored normal life span. The rescue was not due to the tip itself because an identical tip filled with dry cotton wool had no effect. Furthermore, addition of a tip containing wet cotton wool also restored normal life span. This was reproducible when the food was made from a different yeast [SYBaker's (2)] with salt added (Table 1, experiment 2). Thus, our technique is effective in delivering water to salt-stressed flies and in rescuing the associated shortening of the life span. Table 1. Providing a water source to flies rescues the life-shortening effect caused by addition of salt (NaCl) to food, but not life-span alterations due to dietary restriction Next, we established that the life-span change associated with DR was not rescued by water addition [Table 1, experiment 3; replicate of our published data (2)].These data demonstrate that DR in Drosophila under our conditions is not due to rescue of hydration stress. These results directly contradict data from an unreplicated experiment by Ja et al. using conditions ostensibly replicating ours, which reported that life-span extension by DR was eliminated by the addition of water, concluding that hydration stress explains DR (figure 2, G–I, in ref. 4). There are two problems with this conclusion. First, Ja et al. used more sugar (100 g·L−1) in their concentrated medium (CM) than did we, and this higher concentration causes a significant reduction in egg laying compared with the level we use (50 g·L−1) (2), perhaps because of water stress. [Furthermore, fly feeding behavior can be dramatically reduced as sugar concentration increases in this range, which may explain why Ja et al. observed lowered feeding in their CM (figure 1F in ref. 4) and we do not.] Second, the data in figure 2, G–I, from Ja et al. (4) demonstrate that water addition shortened DR life span to the level of the CM (Fig. 1). Thus the “rescue” of the DR effect by water addition could also be explained by water shortening the life span of DR flies, rather than increasing the life span of those on CM. Fig. 1. Life-span differences due to DR and water addition. Ja et al. (4) reported significant extension of life span when the concentration of nutrients in a Brewer's yeast diet were diluted (DR vs. CM) and that this difference was eliminated by the addition ... These data presented from our laboratory, both here and elsewhere (2, 3), robustly demonstrate that hydration stress does not explain DR under the conditions that we use. Furthermore, we have discovered that DR in Drosophila is mediated by an amino acid imbalance in the food that is not modified by water addition (3).

A Modified Excised Leaf Disc Method to Estimate the Toxicity of Slow- and Fast-Acting Reduced-Risk Acaricides to Mites

The "modified excised leaf disc method" is based on leaf discs that fit tightly the bottom halves of 50-mm petri dishes. The bottom half of each petri dish is covered with wet cotton wool to prolong leaf freshness. The side wall of each bottom half has a small hole to allow the petiole of the leaf disc to protrude outside the petri dish. The top half of each petri dish has a 28-mm (diameter) window. For phytophagous mites the window is covered with a 40-microm mesh Pecap polyester screen. Using this method it was possible to estimate the LC50 value of bifenazate to be 0.00413 g (AI)/liter and the LC50 value of spirodiclofen to be 0.40050 g (AI)/liter to the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). The method reduces losses due to escapees and allows observations to be made as long as 9 d after treatment.

First Report of Raceme Blight Caused by Cladosporium cladosporioides on Macadamia Nuts in South Africa.

In September of 2005 and 2006, macadamia (Macadamia integrifolia Maiden & Betche) orchards in Tzaneen, Modjadji, Politsi, and Levubu in the Northern Province and Kiepersol in Mpumalanga, South Africa were surveyed and sampled to determine the causal agent of raceme blight. Symptoms appeared during early bloom and were present on racemes of all developmental stages. Early signs were necrotic tips of the peduncle that often curved to one side with necrosis spreading upward, resulting in the so-called "rat tail". Unopened flowers were also affected. In severe cases, the entire inflorescence (flowers and peduncle) was necrotic and eventually flowers abscised. Occasionally, infection began with single flowers as small water-soaked specks on the flower, with no symptoms on the green peduncle. Diseased racemes were covered with olive gray patches of mycelia and abundant conidia. Flowers with blight symptoms were collected, surface disinfested with 70% ethanol for 2 min, and left to dry. Thirty isolations were made from the interface of the lesion and healthy tissue, plated onto 50% potato dextrose agar (PDA) (Biolab, Merck Laboratories, Wadeville, South Africa) with 19 g of agar per liter, and incubated at 25°C for 5 days. Direct isolations from diseased material were done by picking up conidia and placing them on PDA. A fungus was isolated consistently and identified morphologically as Cladosporium cladosporioides (Fresen.) de Vries based on the velvety olive-brown with almost black reverse colony color and dimensions and color of conidia and conidiophores. Conidia formed in long branched chains that readily disarticulate, mostly aseptate, elliptical to limoniform, 3 to 10.5 (3 to 7) × 2 to 5 (3 to 4) μm. Conidia were pale to olive brown and smooth to verruculose. Ramoconidia were 0-1 septate, 2.5 to 5 μm wide, up to 28 μm long, smooth or sometimes minutely verruculose. Conidiophores were pale to olive brown, macro- and micronemateus, smooth or sometimes verruculose, and of various lengths up to 320 μm long and 2 to 6 μm wide. To confirm pathogen identity, the ITS 1 and ITS 4 regions were sequenced, which had 100% homology to the 18S rRNA of C. cladosporioides (GenBank Accession No. DQ 124142.1). Pathogenicity trials were conducted in the field. Fungal isolates were grown on PDA for 6 days, spores were harvested, and a suspension was made (106 spores ml-1). Twenty macadamia inflorescences (cv. Beaumont) were dipped in the suspension for 1 s, covered with plastic bags containing wet cotton wool, and covered with paper bags. Inflorescences in different stages (petal fall, knee stage, and closed) were inoculated. Control treatments were dipped in sterile water. After 2 to 3 days, the bags were removed. Symptoms developed on all 20 inflorescences and in all cases, the bottom of the inflorescence blighted, resulting in the typical rat tail symptom. C. cladosporioides was reisolated from all surface-disinfested infected material plated on PDA. Control inflorescences developed no symptoms. Isolate PPRI 8376 was deposited with the National Collection of Fungi, Plant Protection Research Institute, Pretoria, South Africa. The disease is prevalent during wet periods and 5 to 10% of flowers were infected. The disease has increasingly been seen in orchards over the last two seasons and under favorable wet, humid conditions, severe infections have resulted in 100% flower loss. To our knowledge, this is the first report of C. cladosporioides causing raceme blight on macadamia in South Africa.

First Report of Phomopsis longicolla on Cocklebur (Xanthium strumarium) in Croatia.

Cocklebur (Xanthium strumarium L.; family Asteraceae) is a widespread weed species in eastern Croatia found especially in arable crops including soybean. Symptoms of disease appear after the plants reach physiological maturity. Stems and branches are completely blighted, and on the surface, are covered with minute, black pycnidia embedded in the epidermal tissue of the host and are especially numerous around nodes. More than 100 plants with symptoms were examined. From each plant with symptoms, three pieces of symptomatic tissues (5 to 10 mm) were disinfected and placed on potato dextrose agar (PDA), pH 4.5 and 25°C with a 12/12 h of light/dark regimen. The cultural and morphological characteristics of the fungi isolated from X. strumarium corresponded with those described (1) for Phomopsis longicolla Hobbs isolated from soybean. P. longicolla frequency was 3%, while other isolates belonged to other Phomopsis species. To confirm the morphological identification of isolates, molecular identification was performed. DNA of four isolates was extracted from 7-day-old monoconidial cultures grown on PDA. The internal transcribed spacer (ITS) regions of ribosomal DNA were amplified with universal primer ITS4 and ITS5 and sequenced (M-Medical Genenco, Rome). The sequences were aligned with the multiple sequence alignment program ClustalW, showing 100% similarity among them, and a sequence (GenBank Accession No. EF026104) was compared with the ITS sequences available on the database, revealing that it is identical to many P. longicolla isolates. To confirm Koch's postulate, cocklebur plants were infected in the field by applying mycelial plugs (5 mm in diameter) from the margin of 6-day-old cultures to the plant stem. The inoculation point was internodal at the mid-stem. After inoculation, plugs were covered with a piece of cotton wool and aluminum foil. Stem lesions were measured 10 days after inoculation. Mean stem lesions were 15 to 21 mm. A pathogenicity test was also done on soybean cv. Tisa (21-day-old) seedlings by applying mycelium plugs (5 mm) with a sterile scalpel on previously wounded hypocotyls. The inoculate point was covered with wet cotton wool and aluminum foil. After 10 days, mean stem lesions were 18 to 30 mm. The pathogen was always reisolated from the stem lesions. Control plants inoculated by PDA plugs did not exhibit any symptoms. There is a report of P. longicolla on cocklebur in the United States (2) and on other plants from the Asteraceae family. Other weeds such as Abutilon theophrasti Med., were shown to be a host of fungal pathogens belonging to the Phomopsis/Diaporthe complex of soybean (3). Our results also confirm that cocklebur could be a natural inoculum source for Phomopsis seed decay of soybean caused primarily by P. longicolla. However, to our knowledge, this is the first report of P. longicolla being isolated from naturally infected cocklebur in Croatia.

The potential of some spice essential oils in the control of A. parasiticus CFR 223 and aflatoxin production

Essential oils of sweet basil ( Ocimum basilicum), cassia ( Cinnamomum cassia), coriander ( Coriandrum sativum) and bay leaf ( Laurus nobilis) at 1–5% (v/v) concentration in palm kernel broth inoculated with spore suspension (10 6/ml) of Aspergillus parasiticus CFR 223 were evaluated for their potential in the control of aflatoxigenic fungus A. parasiticus CFR 223 and aflatoxin production. Healthy sorghum grains (120/treatment) immersed in the oils and distributed in three petri dishes with wet cotton wool were also inoculated with spore suspension (10 6/ml) of A. parasiticus CFR 223 and assayed for grain protection. Sweet basil oil at optimal protective dosage of 5% (v/v) was fungistatic on A. parasiticus CFR 223 and aflatoxins produced in vitro and on fungal development on sorghum grains ( P ⩽ 0.05) with a residual effect that lasted for 32 days. In contrast, oils of cassia and bay leaf stimulated the mycelia growth of the fungus in vitro but reduced the aflatoxin concentration (B 1 + G 1) of the fungus by 97.92% and 55.21% respectively, while coriander oil did not have any effect on both the mycelia growth and aflatoxin content of the fungus. The combination of cassia and sweet basil oils at half their optimal protective dosages (2.5% v/v) completely inhibited the growth of the fungus. The feasibility of implementing the results of this study to control aflatoxins was examined by the addition of whole and ground dry basil leaves at 5% and 10% (w/w), respectively, to 10 g sorghum, groundnut, maize and melon seed after 35 days storage period. It was found that the addition of whole and ground basil leaves markedly reduced aflatoxin contamination; however, 10% (w/w) of whole leaves was more effective as the reduction in aflatoxin was between 89.05% and 91%. The findings showed that aflatoxins can be controlled by co-storing whole sweet basil leaves with aflatoxin infected foods. The economic value of the study lies in the simplified technique for control of aflatoxin contamination in agricultural products and the benefits derivable from the use of local resources.

Isolation of Diaporthe phaseolorum var. caulivora from Abutilon theophrasti in Croatia

During 2000–03 a weed survey was carried out in fields of soybean, sunflower and sugar beet in Croatia. Abutilon theophrasti (velvetleaf), found in soybean fields, showed symptoms of black lesions, several centimetres in length, on the stems and side branches. A fungus was isolated from stem lesions on potato dextrose agar (PDA) and incubated at 25°C, with a 12 h dark/light regime. On PDA the fungus quickly formed a dense white mycelium, which later became fluffy. The mycelium was yellow-white at first, turning yellow-brown later. After 10 days black stromata were produced. These were irregularly shaped and measured 2–5 mm in diameter. Numerous black perithecia were formed within the stromata. Ten perithecia, 50 asci and 100 ascospores were measured. The sizes of perithecia were 200–360 × 295–450 µm and these had a protruding neck which was highly variable in length (300–1200 µm). The sizes of the asci were 25·1–42·0 × 4·2–8·3 µm. The ascospores were two-celled, hyaline, elongate to ellipsoidal, biguttulate, constricted at the septum, and measured 8·0–13·1 × 2·5–4·4 µm. Based on the morphological characteristics, the pathogen was identified as Diaporthe phaseolorum var. caulivora. Velvetleaf plants were infected in the field by applying a mycelium plug to the plant stem. The inoculation point was covered with a piece of wet cotton wool and aluminium foil to retain moisture. Lesions 5–19 mm long were observed 10 days postinfection. Diaporthe phaseolorum var. caulivora was reisolated out of the lesions of inoculated plants, fulfilling Koch's postulates. A pathogenicity test was also done on soybean seedlings. Mycelium plugs were put on the hypocotyl, which was previously wounded with a sterile scalpel. After 10 days the pathogen was reisolated from the stem lesion of inoculated plants. Uninfected control plants did not exhibit any symptoms. To confirm pathogen identification, molecular analysis was performed. DNA was extracted from a monoconidial culture and amplified with ITS4 and ITS5 universal primers (White et al., 1990). The ITS was sequenced and submitted to GenBank (Accession No. AY857867). Comparison of the ITS sequences available on the database revealed that it was almost identical, with only two deletions in positions 24 and 542, to D. phaseolorum var. caulivora (AJ312360). It is known that weeds can act as a reservoir for infection by D. phaseolorum (Black et al., 1996). Abutilon theophrasti has been recorded before as a host for D. phaseolorum var. sojae (Hepperly et al., 1980) and Phomopsis longicolla (Li et al., 2001). Each year in Croatia the presence of fungi in the Diaporthe/Phomopsis complex can be observed on soybean, but these fungi do not appear to cause great economic damage under local conditions. This is, however, the first report of A. theophrasti as a host for D. phaseolorum var. caulivora in Croatia.

A new, rapid and non-invasive technique to inoculate plants with Phytophthora cinnamomi

A simple, effective method of inoculating Eucalyptus marginata (jarrah) stems with the pathogen Phytophthora cinnamomi was developed. There were four criteria to be addressed when developing this technique. A method was required that did not necessitate the wounding of plant tissue, that simulated surface water ponding (often seen in riplines on rehabilitated mine sites), that was applicable to both glasshouse trials and field trials, and that was less labour intensive than previously published methods. In the development of this new method in the glasshouse, agar plugs, colonised by P. cinnamomi, were placed face-down onto unwounded green stem and periderm of 18-month-old jarrah seedlings. The highest percentage of stems infected was obtained when stems were pre-moistened before the inoculum plug was held in place with wet cotton wool and Parafilm. This technique is simple to use under field conditions.

Induced defence in detached uninfested plant leaves: effects on behaviour of herbivores and their predators.

Induction of plant defence against herbivores may include the attraction by volatile infochemicals of natural enemies of the herbivore. The emitted volatiles that mediate this attraction may also affect the behaviour of the herbivore itself. In this paper we investigate the response of the herbivorous spider miteTetranychus urticae and the predatory mitePhytoseiulus persimilis towards volatiles whose production is induced in detached Lima bean leaves. Detached uninfested Lima bean leaves were incubated on wet cotton wool on which bean leaves infested with spider mites (T. urticae) were present simultaneously or had been present previously. These treatments induce the production of volatile infochemicals in the uninfested bean leaf tissue: predatory mites are attracted and spider mites are deterred. These are the first data on the response of predators and herbivores to plant volatiles whose production was induced in detached uninfested leaves.

Induction of indirect defence aganist spider-mites in uninfested lima bean leaves

Headspace analyses of uninfested Lima bean ( Phaseolus lunatus) leaves show an absence of or only trace amounts of the terpenoids ( E)-β-ocimene and ( E)-4,8-dimethyl-1,3,7-nonatriene. Upon infestation by two-spotted spider-mites ( Tetranychus urticae), Lima bean leaves produce ( E)-β-ocimene and ( E)-4,8-dimethyl-1,3,7-nonatriene which attract predators of the herbivore, a phenomenon known as indirect defence. When uninfested Lima bean leaves were placed on wet cotton wool upon which leaves that were partially infested with spider-mites, were also placed, the uninfested leaf tissue emitted the terpenoids in relatively high amounts: ca 20–40% of the amounts emitted by the infested leaf tissue. The same effect was found when uninfested leaves were put on wet cotton wool on which infested leaves had previously lain. The data show that an elicitor(s) from spider-mite infested leaves induces the production of ( E)-β-ocimene and ( E)-4,8-dimethyl-1,3,7-nonatriene in uninfested Lima bean leaves. This is the first chemical evidence for induction of indirect defence in uninfested leaves.