Red clover (Trifolium pratense L.) is characterized by high dry matter (DM) yield and a high nutritive value. It has a higher concentration of soluble carbohydrate than lucerne and its protein is less degradable in the rumen than lucerne protein. The aim of this study was to quantify the main crude protein (CP) and carbohydrate (CHO) fractions of a tetraploid (4n, cv K-32) and a diploid (2n, cv K-39) red clover cultivar harvested at three stages of development in the spring growth. Results showed that the cultivar and stage of development had a large impact on chemical composition and the protein and carbohydrate fractions determined by the Cornell Net Carbohydrate and Protein System (CNCPS). The rapidly degradable protein fraction (PA) was significantly higher in the tetraploid (272 to 287 g kg-1 CP with advancing plant development) than in the diploid red clover cultivar (214 to 268 g kg-1 CP). Rumen degradable carbohydrate concentration was not affected by cultivar but it was significantly influenced by the stage of development. The concentration of rumen degradable protein decreased from 726 to 655 g kg-1 CP with advancing plant development in the diploid red clover cultivar. In conclusion, the tetraploid red clover cultivar was higher in CP, its protein was more rapidly degradable, and it was characterized by lower concentrations of lignin and unavailable carbohydrate fractions than the diploid cultivar.

Penicillium crustosum Thom. is a fungus commonly found on cheese and nuts, but is also a postharvest pathogen that causes blue mold disease of pome and stone fruits including plum and nectarine (Louw and Korsten 2016; Restuccia et al. 2006). The fungus produces mycotoxins (penitrem A, roquefortine C, terrestric acid, and cyclopenol) which are of concern for human health (Frisvad and Samson 2004). In Serbia, P. crustosum has been previously described on apple fruit (Vico et al. 2014). On nectarine fruit (Prunus persica var. nucipersica), after 6 weeks of cold storage, symptoms of blue mold developed in a fruit market in Belgrade, Serbia. The fruit was collected and isolations performed in November 2017. Decayed areas on infected fruit were soft, light to medium brown with blue-green sporulation on the fruit surface. Two isolates were obtained (N2AS and N2BS) and cultured on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), yeast extract sucrose agar (YES) and potato dextrose agar (PDA) at 25°C for 7 days. Isolates were identified as P. crustosum based on morphological features (Frisvad and Samson 2004; Pitt and Hocking 2009). On all media, mycelia were white and colonies turned blue-green with abundant sporulation. Colonies of both isolates were radially sulcate on MEA and YES, and plane with a granular texture on CYA and PDA, and were yellow to orange on the reverse side on YES. Mean colony diameter on PDA was 29.2 ± 1.2 mm for N2AS, and 31.3 ± 1.4 mm for N2BS; on CYA 30.8 ± 1.2 mm for N2AS and 30.9 ± 1.1 mm for N2BS; on YES 40.7 ± 3.6 mm for N2AS and 43.6 ± 1.4 mm for N2BS; and on MEA 33.4 ± 1.2 mm for N2AS and 34 ± 2.5 mm for N2BS. Crusts of conidial masses formed on MEA and PDA after 10 days. Conidiophores of both isolates were terverticillate, stipes were septate with rough walls, and conidia, borne in columns, were smooth and spherical to subglobose. Conidial diameter for N2AS was 2.32 to 3.95 (average 3.13) µm and for N2BS was 2.34 to 3.98 (average 3.27) µm (n=50). Isolates formed a yellow ring, using Ehrlich’s reagent, indicating lack of cyclopiazonic acid, but production of other alkaloids. Morphological identification was confirmed by isolating genomic DNA, PCR amplification of the partial β-tubulin gene using Bt2a/Bt2b (Glass and Donaldson 1995) and sequencing. BLAST analysis revealed that N2AS sequence (MT799805) was 99% similar and N2BS (MT799806) was identical to sequences AY674351 (strain CBS 101025) and KJ775121 (strain DTO_244H8) of P. crustosum in GenBank. Sequences (2X consensus) of the two isolates differed in one nucleotide showing the existence of single-nucleotide polymorphism among P. crustosum isolates. Pathogenicity was tested on nectarine, peach and apple fruit (four fruit per isolate and the control). Fruit were washed, surface-sanitized with 70% ethanol, and wound (10x4 mm) inoculated on two sides with 40 μl of a 105/ml conidial suspension in sterile distilled water containing 0.1% Tween 20 (TSDW). Control fruit was inoculated with TSDW. Inoculated and control fruit were stored at 25°C for 7 days. Inoculated fruit developed light brown decay with cracks in the epidermis that spread from the inoculation point on nectarines and peaches. Blue-green sporulation was present on all inoculated fruit. Control fruit remained symptomless. The fungus was re-isolated and was morphologically identical to the original isolates, thus completing Koch's postulates. This is the first report of P. crustosum causing postharvest blue mold decay on nectarine fruit in Serbia. Results show that P. crustosum is not only present as a postharvest pathogen of apple fruit, but of nectarine as well and may pose a threat in storage of both pome and stone fruits in Serbia. References: Frisvad, J. C. and Samson, R. A. 2004. Stud. Mycol. 49:1. Glass, N.L. and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61: 1323. Louw, J.P., and Korsten, L. 2016. Eur. J. Plant Pathol. 146: 779. Pitt, J. I. and Hocking, A. D. 2009. Fungi and food spoilage, 239. Springer. Restuccia et al. 2006. J. Food Prot. 69: 2465. Vico, I., et al. 2014. Plant Dis. 98:1430. Acknowledgment: This research was supported by the project III46008, No. 451-03-68/2020-14/200116, financed by the Ministry of Education, Science and Technological Development, Republic of Serbia.

This study presents the results of testing the effect of different concentrations of lead-acetate on seed germination and young Seedlings of alfalfa. Тhe experiment was organized so that in the first variant, only lead-acetate of different concentrations (10-5, 10-4, 10-3, 10-2 and 2 x 10-2 M) was used, and in the second, next to the lead acetate EDTA concentration of 0.012 % was added. The results of this study in which natural conditions are imitated showed that the percentage of alfalfa seed germination decreases with increasing lead-acetate concentrations. In a medium of high concentration of lead acetate 10-2 and 2 x 10-2, a small number of seeds (39% and 32 (32%) germinated. In the same treatments with EDTA, the toxic effects of lead acetate were reduced, and seed germination was better (59% and 43% seeds germinated). It has been noticed that lead has toxic effects on the growth of alfalfa roots and stems. In the variant in which EDTA was used, the negative influence of lead on and growth of alfalfa seedlings was significantly mitigated.

This study presents the results of testing the effect of different concentrations of lead-acetate on seed germination and young Seedlings of alfalfa. Тhe experiment was organized so that in the first variant, only lead-acetate of different concentrations (10-5, 10-4, 10-3, 10-2 and 2 x 10-2 M) was used, and in the second, next to the lead acetate EDTA concentration of 0.012 % was added. The results of this study in which natural conditions are imitated showed that the percentage of alfalfa seed germination decreases with increasing lead-acetate concentrations. In a medium of high concentration of lead acetate 10-2 and 2 x 10-2, a small number of seeds (39% and 32 (32%) germinated. In the same treatments with EDTA, the toxic effects of lead acetate were reduced, and seed germination was better (59% and 43% seeds germinated). It has been noticed that lead has toxic effects on the growth of alfalfa roots and stems. In the variant in which EDTA was used, the negative influence of lead on and growth of alfalfa seedlings was significantly mitigated.