Evaluation of Growth Characteristics of Four Clover Species (Trifoliums pp.) Affected by Freezing Stress under Controlled Conditions

In Iran, clover is the second most important forage crop after alfalfa (Medicago sativa) and is cultivated on a surface of about 70,000 hectares. Yield improvement of this plant could be achieved through autumn cultivation. Iran is still one of the main centers for the diversity of clover species, there is not much information about freezing tolerance of native species of this important plant. Since freezing stress is one of the most important winter stresses which causes severe damage to the plant cells and tissues and ultimately plant death, evaluation of freezing tolerance of this crop could help to determination of the best planting date in order to achieve the highest yield. The aim of this study was to evaluate the freezing tolerance of four clover species through assessment of plant survival and re-growth after freezing stress.

In order to study growth criteria of four clover species after freezing stress in controlled conditions, an experiment was conducted in 2012-13 in Faculty of agriculture, Ferdowsi University of Mashhad. Treatments were arranged as factorial based on Completely Randomized Design with four replications. Factors were included of planting dates (late September and late October), clover species (red clover T. pratense, white clover T. repense, Persian clover T. resupinatum (autumn species) and crimson clover T. incarnatum (spring species) and freezing temperatures (0 (control), -3, -6, -9, -12, -15, -18, -21ºC). Survival percentage, LT50 according to Survival (LT50su), leaf area, Reduced 50% Leaf Area Temperature (RLAT50), dry weight and Reduced Dry Matter Temperature 50 (RDMT50) were measured after three weeks re-growth.

Results indicated that survival percentage was significantly affected by interaction of planting date, species and freezing temperatures. In both planting dates, crimson and white species showed a good survival till -15°C while they were killed as temperature was more decreased to -18°C. In first planting date, two species of red and Persian clover had 50 and 42% survival, but were killed in this temperature in the second planting date. The lowest LT50su in autumn (red, white and Persian) and spring (crimson) species was found in first and second planting dates, respectively indicating higher freezing tolerance in crimson species at second planting date and a better acclimation of autumn species at first planting date which led to a higher survival compared to spring species. Leaf area was significantly affected by planting date, species and freezing temperatures at the end of the recovery period. In first planting date, decreasing temperature from zero to -18°C caused reduction of 98 and 48% of leaf area in red and Persian species while no plant of white and crimson species was survived in this condition. In second planting date, leaf area was decreased about 6, 33 and 2% as temperature decreased from zero to -15°C while leaf area of Persian species, not only was not decreased, but also increased by about 12%. The lowest and highest RLAT50 were observed in Persian and red species. Interaction of planting date and species on RDMT50 was significant in a way that in delayed planting date, RDMT50 was increased in red, Persian and crimson species while it was decreased in white species.

Red and Persian species showed higher freezing tolerance according to indices of survival percentage and LT50su. The highest recovery ability of plants based on leaf area per plant, RLAT50, plant dry matter and RDMT50 was found for second planting date. Also annual species of Persian and crimson had lower RLAT50 and RDMT50 and higher freezing tolerance compared to perennial species of red and white clover.