Authors: Madhubala Thakre, K. Prasad and M. K. Verma

Frost is one of the serious abiotic stresses which affect several fruit crops of temperate, tropical and subtropical nature. However, the extent and nature of damage varies among various fruit crops. Frost injury is mainly due to ice formation, whether, it is inter-cellular or intra-cellular. In case of intracellular ice formation, ice forms inside the cell and disrupt its protoplasmic structure mechanically. Although, the duration of freezing does not affect the level of injury. But, the speed of drop in temperature and the level of super cooling affect the extent of damage. In case of intercellular freezing, the ice crystal forms outside the cells i.e. in between the spaces available among the cells. As ice starts to form in intercellular spaces, it favours withdrawal of water more from the neighbouring cells. It actually happens due to the fact that vapour pressure is lower over ice (which is present in intercellular space) than over water available in liquid form (inside the cell). It ultimately creates a gradient of vapour pressure by virtue of which water come out from the cell through semi-permeable cell membrane thus balancing difference in vapour pressure. This dehydration of cells results in more solute concentration inside the cell which stops further cell dehydration by retaining water more tenaciously (by protein, sugars and other molecules). Cell dehydration results in the contraction of the cell wall and the protoplast. It is different from the true plasmolysis, in which only protoplasm contraction occurs, receding from the cell wall. Whereas, in case of frost injury, the cell wall expands upon thawing, but the protoplast remains contracted. This is due to the inability of dead protoplast to reabsorb water. This phenomenon is known as ‘frost plasmolysis’. Uninjured cells are able to reabsorb the water and return to a normal appearance, whereas, injured cells always lose their semi-permeability.

Frost injury is due to ice formation then it means that it will be more in those plant parts which are more succulent as they are having more water content. But, there is one more factor which catalyzes ice formation at comparatively warmer temperature leading to increased susceptibility of plant towards frost injury. This factor is ice nucleation bacteria.


Ice nucleation bacteria belong to heterogeneous ice nuclei class. Ice nuclei are catalysts that are responsible for the water-ice phase transition i.e. ice formation. Ice nuclei have two classes: homogenous and heterogeneous. Homogenous ice nuclei are responsible for catalyzing ice formation at lower temperature. Whereas, heterogeneous ice nuclei catalyzes ice formation at comparatively higher temperatures approaching 0°C. Some non-biological source of heterogeneous ice nuclei are silver iodide, steroids, amino acids, protein, terpines, metaldehyde, a-phenazine etc.

Bacterial ice nucleation activity is limited to some gram negative bacteria. There ubiquitous presence on plants and in other natural habitat makes bacterial ice nucleation a common phenomenon in nature. The phenomenon of bacterial ice nucleation was first observed in strains ofPsuedomonas syringae in 1974 (Maki et al., 1974),thereafter strains of Erwinia herbicola (Lindow et al., 1978), Psuedomonas fluorescens (Maki and Willoughby, 1978), Psuedomonas viridiflava (Paulin and Luisetti, 1978) and Xanthoonas campestris pathovar translucens (Lim et al., 1987) were also reported to have the ability to catalyze ice formation in supercooled water (water cooled below its freezing point without solidification or crystallisation).


The INA bacteria lower the supercooling ability of plants. It means that if a plant can supercool at -10°C in the absence of INA bacteria (water will remain in liquid phase). Then, if INA bacteria will present, ice will form at comparatively warmer temperature. These bacteria present in large epiphytically population on plant surfaces, they reduces the supercooling ability of the plant. As a result ice will form on or in the plants at comparatively warmer temperature (above -5°C); temperature exists mostly at the time of frost event. The maximum population of ice nucleation active bacteria ranged from approximately 100 cells / g fresh weight of Valencia and Navel orange (Citrus spp.) leaf tissue to over 107cells / g fresh weight on leaves of English walnut (Jugans regia L.) and Almond (Prunus amygdalus L.). Large epiphytic population of ice nucleation active bacteria (principally P. syringae) are present on emerging flowers and leaves of these plants. Psuedomonas syringae and Erwinia herbicola, these are two important species active in ice nucleation. Other organic and inorganic material such as dust particles also nucleates ice, only at temperatures lower than -10°C. Thus, it shows the importance and sole involvement of INA bacteria in frost injury.


In all ice nucleating bacteria from nearly all ice nucleating species, bacterial ice nucleation is conferred by single genes. Although ice nucleation genes vary slightly in length, their overall structure is similar. All the genes encode a protein with small unique amino and carboxyl termini but with a similar randomly repeated16 amino acid motif in the internal region that encompasses a large fraction of the protein (Wolber, 1992 and Warren, 1987).


Strategies responsible for reducing the population of INA bacteria over plants can be used for reducing the frost injury. This can be done by applying bactericide to kill bacteria. Application of antagonistic bacteria is another way. Application of ice nucleation inhibitors such as extremes of pH; specific heavy metals ions etc. are also can be used to manage frost injury.


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About Author / Additional Info:
Scientist, Division of Fruits and Horticultural Technology, IARI, New Delhi.