Haploid Embryogenesis in Isolated Microspore Culture of Carrots (Daucus carota L.)

The process of embryogenesis in isolated microspore culture was studied in eight carrot accessions of different origin. The ½NLN-13 medium supplemented with 0.2 mg/L 2,4D and 0.2mg/L kinetin was used to induce embryogenesis. The temperature treatment was performed at 5–6 °C for three days, followed by cultivation at 25 °C in darkness. As was shown, the first embryogenesis was only observed in microspores at the late vacuolated stage when the nucleus moved from the center to one pole following the long cell axis. Depending on the nucleus position, the microspore can divide into two equal or two different sized cells. Following divisions occurred either in one of these cells or in two. However, microspores that divided into two unequal cells were morphologically different form bi-cellular pollen grain. Embryogenic divisions in bi-cellular pollen grains were not observed. First divisions began by the third day of cultivation, and continued until the globular embryoid stage that was well-seen after the fourth week of cultivation. The already-formed embryoids can develop the secondary embryoids on their surface. Depending on the genotype, up to 1000 secondary embryoids can be produced from one embryoid in the liquid MSm medium supplemented with 0.1 mg/L of kinetin for regeneration. All carrot accessions studied were split into three groups: responsive genotypes, weakly responsive genotypes, and reluctant genotypes. The highest yield was 53 initial embryoids per a 6 cm diameter petri dish. Thus, the Nantskaya 4 cultivar totally produced 256 initial embryoids, out of which 94 developed into green plantlets and 162 into albino plantlets, whereas 97 initial embryoids with 45 albino plantlets formed from them were obtained from Chantenay cultivar.

Storage Rot of Dragon Fruit Caused by Gilbertella persicaria

In October 2011, a new disease of dragon fruit (Hylocereus costaricensis) was discovered in a fruit market in Yuanjiang, Yunnan Province, China. Small, light brown, water-soaked spots appeared initially and then coalesced, extending to the entire fruit in 6 days. Hyaline hyphae and light brown sporangia were observed over the entire surface of the infected fruit. On potato sucrose agar (PSA) the fungus produced a white, appressed colony that covered a 9-cm diameter petri dish in less than 5 days at 25°C. The sporangiophores were hyaline, light brown to grayish, 44.71 to 143.14 (average = 85.10) μm long, and arose directly from the non-septate substrate hyphae. The sporangia were spherical, single, and terminal and yellow-brown to brown when young turning to dark brown or black at maturity. Both the sporangiophores and sporangia were covered with calcium oxalate crystals. When mounted in a drop of water, the sporangium immediately broke longitudinally into two halves, releasing the spores and exposing a large pyriform columella at the tip of the sporangiophore. The spores were mostly globose to ellipsoid, aseptate, and 5.15 (3.71 to 7.86) × 6.30 (4.08 to 9.19) μm (n = 300). Two to three slender, hyaline appendages were attached to the ends of the spores. The cardinal growth temperatures of the pathogen were 10, 30, and 40°C and it grew faster in the dark than under 12-h alternating light-dark cycles. The fungus was identified as Gilbertella persicaria (1). To confirm the identification, the internal transcribed spacer region of the nuclear rDNA of one isolate was amplified using the fungal primers ITS1 and ITS4. The nucleotide sequence (Accession No. JQ951601) showed 98% homology with G. persicaria in GenBank (HM999958). Pathogenicity tests were carried out on two species of dragon fruit, H. costaricensis and H. undatus, by placing a 6-mm diameter young mycelial PSA agar disc on the surface of an asymptomatic fruit, either unwounded or wounded with a sterile needle. As the control, a plain PSA disc was used. Each inoculated fruit was placed in a moist chamber and incubated at 25°C. Three fruits were used per treatment and the experiment was repeated twice. The fruits rotted in 2 to 3 days, and the disease was especially serious on wounded fruits and on H. costaricensis. The fungus was reisolated from infected fruits. The controls did not show any disease symptoms. Inoculation studies were also made on other fruits but rot was produced only on peach, pear, and wounded tomato. To our knowledge, this is the first record of dragon fruit rot caused by G. persicaria. The fungus had been reported in China but caused no diseases (2). In India, it caused fruit rot of pear, tomato, and peach (3). To minimize the disease, dragon fruit should be stored at low temperature and in uncovered containers. References: (1) G. L. Benny. Mycologia 83:150, 1991. (2) J. Y. Cheng and H. Y. Mei. Acta Phytotax. Sin. 10:105, 1965. (3) M. D. Mehrotra. Mycopath. Mycol. Appl. 29:151, 1966.

First Report of Resistance to Benomyl Fungicide in Sclerotinia sclerotiorum

Benomyl fungicide (Benlate) is used worldwide to control ascomycete pathogens, but resistance has developed in several pathogen populations (1). On the Canadian prairies, benomyl is used to reduce injury caused by Sclerotinia sclerotiorum (Lib.) de Bary on canola (Brassica napus, B. rapa) and alfalfa (Medicago sativa) seed crops. To determine if populations are resistant to benomyl, isolates of S. sclerotiorum collected from 15 fields (12 alfalfa and 3 canola, one isolate per field) in 2000 were grown on potato dextrose agar amended with benomyl at 0, 0.05, 0.5, 5, 50, and 500 mg/liter. Plugs of mycelium from the margin of an actively growing colony were placed in the center of a 10-cm-diameter petri dish containing 15 ml of test medium and incubated on a laboratory bench. Linear growth (mean of maximum width and right angle) of each colony (three replicates each) was measured after 5 to 6 days. The growth of isolates from 13 fields was inhibited by low concentrations of benomyl (EC50 < 8 mg/liter), but two isolates were very resistant (EC50 > 200 mg/liter). Resistant cultures were isolated from infected canola plants in the only two fields in the study in which reduced efficacy of benomyl was suspected. The distribution and importance of benomyl-resistant populations of S. sclerotiorum in the region remains to be determined. Reference: (1) T. R. Pettitt et al. Mycol. Res. 97:1172, 1993.

Thin Agar Layer Method for Recovery of Heat-Injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm–diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37°C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55°C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P &gt; 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P &lt; 0.05)—in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.

Effect of Carbon, Nitrogen, and C:N Ratio on Growth, Sporulation, and Biocontrol Efficacy of Talaromyces flavus

Engelkes, C. A., Nuclo, R. L., and Fravel, D. R. 1997. Effect of carbon, nitrogen, and C:N ratio on growth, sporulation, and biocontrol efficacy of Talaromyces flavus. Phytopathology 87:500-505.Five-day biomass production by the biocontrol fungus Talaromyces flavus was measured in a liquid basal medium, pH 5.5, containing each of 37 carbon (C) sources with a single nitrogen (N) source, and each of 42 N sources with a single C source. In general, production of biomass was greatest on complex sugars such as polysaccharides (32 g/liter of medium) and β-glucosides (2.4 g/liter of medium), and was least on monosaccharides (1.3 g/liter of medium). Ascospore production at 6 weeks on solid basal medium with the same amount of these same 37°C sources was greatest on oligosaccharides (2.9 × 108 spores per 5.5-cm-diameter petri dish), and least on polysaccharides and monosaccharides (1.6 and 1.4 × 108 spores per 5.5-cm-diameter petri dish, respectively). For C sources, there was no correlation between production of ascospores and hyphal dry weight. The various N sources yielded 0 to 109 ascospores per 5.5-cm-diameter petri dish and 10-4 to 10-5 g of hyphal dry weight per milliliter. In general, N sources that resulted in the greatest number of ascospores also yielded the greatest hyphal dry weights. For the two C and two N sources tested, the number of ascospores increased as the ratio of C to N increased from 5:1 to 30:1. This effect was most obvious as the C:N ratio increased from 5:1 to 15:1. At low C:N ratios (<15:1), treatments with hypoxanthine as a N source resulted in significantly greater production of biomass than treatments with ammonium tartrate; no difference was observed at C:N ratios ≥15:1. Incidence of Verticillium wilt was 50% lower for eggplants drenched with ascospores grown on potato dextrose agar (PDA) compared with eggplants either nondrenched or drenched with ascospores grown on media with hypoxanthine plus lactose or maltose. Thus, C and N sources that slightly increased ascospore production of T. flavus reduced efficacy of biocontrol of Verticillium wilt compared with ascospores produced on PDA.

Activity of lysosomal cysteine proteinase during differentiation of rat skeletal muscle

Cysteine-proteinase activities were measured in extracts of pre- and post-fusion populations of rat myogenic line L6 cells and in extracts of whole rat muscle. Activities of cathepsins B, L and H were compared. The substrates used included Z-Phe-Arg-NMec (cathepsins B and L), Z-Arg-Arg-NMec (cathepsin B), and Arg-NMec (cathepsin H) (where Z = benzyloxycarbonyl, and NMec = 4-methyl-7-coumarylamide); the enzyme activities were more specifically differentiated by appropriate concentrations of the inhibitors Z-Phe-Phe-CHN2 (CHN2 = diazomethane), bestatin and E-64 [L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane]. These experiments have demonstrated the feasibility of determining the cysteine-proteinase activities of myoblasts from a single (60 mm-diameter) Petri dish, with enzyme concentrations in the range of 5-20 ng/ml. Specific activities of the enzymes in L6 cells increased 2-20-fold after fusion. Concentrations of cysteine proteinases in extracts from cultured myoblasts were two orders of magnitude greater than those in muscle-tissue extracts. Cultured-cell extracts contained endogenous inhibitor(s) to purified rat cathepsins B, L and H.