Plant Disease And Bacterial Soft Rot Biology Essay

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1.1 Plant disease: a threat to global food security

Disastrous plant disease exacerbates the current deficit of food supply in which at least 800 million people are inadequately fed. Plant pathogens are difficult to control because their populations are variable in time, space and genotype. In order to combat the losses they cause, it is necessary to define the problem and seek remedies. At the biological level, the requirements are for the speedy and accurate identification of the causal organism, accurate estimates of the severity of disease and its effect on yield, and identification of its virulence mechanisms. Among the post harvest diseases caused by microorganisms, bacterial soft rot has foremost economic significance.

1.2 Bacterial soft rot: a global problem

Bacterial soft-rot has been considered as one of the most frequent diseases observed in different plant species all over the world and cause great total loss of crops (Agrios, 1997). The disease causes significant economic losses worldwide especially during storage where it spreads from infected host to healthy host (Sharga and Lyon, 1998). Hence, control and management of this disease rely mainly on cultural practices such as planting disease-free seeds and maintaining proper harvesting, handling, and storage conditions. However, in most of the cases, these practices are not sufficient to control the disease effectively.

Bacterial spoilage, in general, is characterized by a watery and slimy appearance. The most common bacterial spoilage of vegetables and fruits is caused by members of the genus Erwinia. This bacterium enters the vegetables through injured leaves and curds. A characteristic of Erwinia spoilage are a water-soaked appearance of the affected tissue, and when the spoilage is well progressed the product is converted into a slimy mass (Ryall & Lipton 1979).

Bacterial soft rot, caused by Erwinia is a devasting post harvest bacterial disease of considerable economic significance, affecting wide varieties of vegetables (van-der Zwet, 1979). The Erwinia are plant pathogens responsible for many $US millions each year in crop losses worldwide (Toth et al 2003). Three soft rot Erwinia, namely Erwinia carotovora subsp. carotovora (reclassified as Pectobacterium carotovorum subsp. carotovorum, Pcc), E. carotovora subsp. atroseptica (reclassified as P. carotovorum subsp. atrosepticum, Pca) and E. chrysanthemi (reclassified as Dickeya chrysanthemum, Dc) are usually associated with this disease (Perombelon and Kelman, 1980).

1.3 Prominent features of the soft rot Erwinia

The plants are composed mainly of polysaccharides (pectate and cellulose; pectate is the main polysaccharide within the middle lamella between plant cells). Plant cell-wall degrading enzymes(PCWDEs) includes pectin methylesterases, pectate lyases, pectin lyases, polygalacturonases, cellulose, proteases and phospholypase that break down these polysaccharides are an essential virulence component of the soft-rot Erwinia (Toth et al., 2003).

The production of PCWDEs from soft rot Erwinia is very tightly regulated process that involves complex regulatory networks of sensor mechanisms that transduce environmental stimuli into gene expression. The PCWDEs and harpins are the major virulence determinants in E. carotovora (Newton and Fray, 2004). Virulence determinants in the soft rot Erwinia are controlled by sometimes interrelated, regulatory networks, which act either positively or negatively on one (targeted regulation) or several (global regulation) determinants. They are stimulated by factors such as oxygen and nitrogen availability, temperature, osmolarity, iron deprivation, growth phase, catabolite repression, plant degradation intermediates, plant extracts, DNA damaging agents and very likely other factors yet to be identified (Hugouvieux-Cotte-Pattat et al., 1996).

The expression of a number of important virulence determinants and secondary metabolites of Erwinia is under Quorum sensing (QS) mechanism control. Erwinia uses QS signal (AHLs) to monitor its cell density and only initiates a pathogenic attack when its population density is above a critical level, which ensures a high probability of overcoming host resistance. AHLs are required to induce the production of secreted plant cell wall-degrading exoenzymes (PCWDEs), synthesis of carbapenum antibiotic, as well as for the expression of harpin genes and components of a type III secretion system (Newton and Fray, 2004).

1.5 Counteracting strategies on bacterial soft rot disease

Any general improvement in agricultural practices can have a tremendous economic impact in which microbiological approach circumscribes all the advancing technical frontiers. A large number of technologies have been developed to harvest the bountiful products that the plants manufacture, including natural dyes, biofertilizers, biocontrol agents and biofuel. Thus, it is imperative to launch a concerted effort by microbiologists, plant scientists and biotechnologist to search for innovative ways to stay one step ahead of natural enemies. Economical, viable and ecofriendly methods of prevention of spoilage by targeting enzymes are required at present. In this context, the search for inhibitors of the important PCWDEs would find a potential use as protection of plant from soft rot infection.

Most spoilage control treatments delay decomposition but will not completely stop it. This disease is controlled mainly with chemicals such as copper-based bactericides and antibiotics (Kyeremeh et al., 2000). Copper treatment has traditionally been considered as bactericidal in agriculture, its effectiveness often being measured by the absence of bacterial growth on a solid medium (Ordax et al., 2006). Its use is one of the most common methods for controlling bacterial plant diseases, but it has led many bacteria to develop different strategies against copper ions (Saxena et al., 2002). Recent studies have shown the induction of the viable-but-nonculturable (VBNC) state by copper in several plant-pathogenic bacteria (Alexander et al., 1999; Grey and Steck, 2001). In this respect, it has been suggested that copper induced VBNC cells of some phytopathogenic bacteria could be related to the persistent nature of infections in copper treated fields (Grey and Steck, 2001). The nature of the VBNC state, however, is still the topic of an intense debate in the literature, and some authors argue that this condition may be a physiological state prior to cell death (MacDougald et al., 1998).

However, the worldwide trend toward ecologically safe methods of protecting crops from pests and pathogens, calls for reducing the use of these chemicals. Biological control is therefore a good alternative. Many researchers have demonstrated the potential of biological control of soft rot of vegetables and fruits (Dong et al., 2002; Dong and Zhang, 2005).

With this background primarily the most predominant strain of Erwinia spp. found in local market fruits and vegetables as well as farming soils needs to be identified so that studies of the main virulence factors i.e. pectinolytic enzymes and a search for biocontrol of the phytopathogen will be facilitated. This information obtained will be beneficial to tackle the problem of post harvest losses in agricultural products to large extent as its long term application. The physiology of Erwinia spp. as regards the pathogenesis and the altered cell physiological state are the aspects that need to be studied in detail to understand the efficacy of the biocontrol measures.

Screening and development of biocontrol agents involving endophytic bacteria against soft rot causing phytopathogenic Erwinia will lead to new approaches for the control as the only current effective controls are through the use of antibiotics or polluting metal based formulas which have significant drawbacks. The research will lead to a reduction in chemicals applied and a significant reduction in crop loss by ecofriendly means.

[1] Studies on soft rot causing phytopathogenic Erwinia

Isolation and characterization of soft rot causing Erwinia.

Polyphasic characterization of virulence properties of soft rot causing bacteria.

[2] Physiological studies with respect to post harvest chemical control agents.

Enumeration and characterization of altered physiological state and survival studies of soft rot causing Erwinia.

[3] Biological control for Soft rot causing phytopathogenic Erwinia.

Development of biocontrol agent using quorum quenching approach.

Labscale application studies.

3.1 Physiological studies with respect to Organic salts, inorganic salts, weak acid and metal compounds.

Different mechanisms have been suggested to explain the antimicrobial effects of the different organic and inorganic compounds, including inhibition of several steps of energy metabolism (benzoate, bicarbonate, propionate and sorbate) (Freese et al., 1973; Wedzicha, 1984; Kabara and Eklund, 1995) and complication to DNA and RNA (magnesium and sodium salts) (Johansons and Wood, 1990). In contrast to inorganic acids, weak acids penetrate bacterial cell membrane as undissociated molecules through permeases and porins and their intracellular dissociating reduces cytoplasmic pH (Phan-Thanh et al., 2000; Jordan et al., 1999). Thus their antimicrobial activity is attributed both to pH gradient across membrane and the reduction in intracellular pH. Also the ability of salts to accumulate the compatible solutes required is reduced in acidic conditions (Farwick et al.,1995; Ogahara et al., 1995).

3.1.1 Effects of compounds on the growth of Pcc.

Effect of 20 different inorganic and organic salts, metal compounds and weak acids on growth of two strains of Pcc cells were analyzed in nutrient broth media amended with particular concentration of the respective salts in four sets inoculated separately with Pcc type strain MTCC1428 and the isolated Pcc strain BR1 cells after 24h.

Among inorganic salts, sodium carbonate, sodium chlorite, and sodium bisulphate shows complete inhibition on the growth of both strains Pcc BR1 and MTCC1428, whereas sodium bicarbonate and tri-potassium phosphate shows 98.62 and 96.18 % growth inhibition respectively of both Pcc strains. Potassium dihydrogen phosphate, di-Sodium hydrogen phosphate, di-Sodium sulphate and amonium sulphate were shown to have significant moderate inhibitory effect on growth of both Pcc strains, which were grouped in group-III (Fig.-1).

In case of organic salts, 0.2M Tri-sodium citrate showed less growth inhibition of both Pcc strains. Almost same level of significant growth inhibition was observed with sodium benzoate, potassium sorbate and sodium propionate at 0.1M concentration for both Pcc strains (group-I). Among organic salts only 0.2M ammonium acetate showed complete growth inhibition for both Pcc strains (Fig.-1).

All three heavy metal salts (group-I) were showed significantly complete growth inhibition of both Pcc strains BR1 and MTCC1428 at very low concentration of 0.001M. Weak acids were also showed similar growth inhibition of both strains at 0.01M concentration, which were grouped in group-II (Fig.-1).

3.1.2. Relationship between pH, osmotic pressure and growth inhibition of Pcc

The acidity or alkalinity of the medium resulting from the addition of the salts to the growth medium can have varying degree of inhibitory effect on the growth of Pcc cells. The pH of the media was varied by addition of the salts at different concentrations.

As shown in Fig.-1, potassium di-hydrogen phosphate, sodium chlorite, sodium bisulphate and three weak acids were strongly acidified the media with ∆ pH ≥ 2 (∆ pH=|7.4 (the optimal pH of medium)-pH of salt amended medium|) whereas sodium carbonate and tri-potassium phosphate made media alkaline with ∆ pH ≥ 3. The growth inhibition was checked by considering pH 7.4 as the optimum pH for the growth of Pcc (Fig.-1).

Two inorganic salts (tri-potassium phosphate and sodium carbonate), three organic salts (potassium dihydrogen phosphate, sodium chlorite and sodium bisulphate), and three weak acids (butyric acid, hexanoic acid and octanoic acid), which were showed high growth inhibition is likely to be associated with high energetic demands posed by extreme pH variations (Shabala et al., 2008) on both Pcc strains, no other growth inhibition pattern can be correlated with the difference in the pH of the amended media (Fig.-1). Ammonium acetate, sodium bicarbonate, ammonium sulphate, tri-sodium citrate, copper sulphate, manganese chloride, lithium acetate and di-Sodium hydrogen phosphate showing variable degree of growth inhibition showed low ∆pH in range of 0.2-1.1, while sodium benzoate, potassium sorbate and sodium propionate with no difference in pH inhibited the growth of Pcc BR1 by 99%, 92% and 99% respectively (Fig.-1). These results were in contrast of earlier observation reported by Samelis et al. (2003), concluded that even the small differences in pH, such as 0.5 units, and may have major impact on the survival of pathogens.

Osmotic pressure of the different salt solutions and weak acids was calculated using vant Hoff's equation, ∏ = iRTc, where R is the gas constant, T is the absolute temperature (K), c is the concentration of the salt (mol/liter), and i is the number of ions into which the salt dissociates in solution (Yaganza et al., 2009). Tri-potassium phosphate and tri-sodium citrate were having osmotic pressure 19.89atm whereas Sodium carbonate, Di-sodium hydrogen phosphate, Di-Sodium sulphate and Ammonium sulphate were having 14.92atm osmotic pressure of their salt solution. All other compounds with 0.2M concentration were having osmotic pressure of 9.95atm of their salt solutions. Organic salts (sodium benzoate, potassium sorbate and sodium propionate) and Inorganic salts (sodium chlorite and sodium bisulphate) were having 4.97atm osmotic pressure. While weak acids and heavy metal compounds were having 0.249 and 0.05 atm osmotic pressure, except for manganese chloride (0.075 atm) (Fig.-1).

These results suggested that osmotic pressure alone may not have brought about the inhibition on growth of both strains of Pcc. There have been suggested elsewhere, that the resistance of bacterial pathogens to acidic or osmotic environments is the outcome of their ability to overcome with the increased homeostatic burden caused either by maintaining intracellular



Fig.-1. Relationship between Osmotic pressure (∏) , pH difference of the medium amended with each compound (∆ pH) = │7.4 [the optimal pH for growth] - the pH of the salt-amended medium│and growth inhibition of [A] Pcc BR1 and [B] Pcc MTCC1428 by 1) 0.2M Sodium carbonate, 2) 0.2M Ammonium acetate, 3) 0.2M Potassium dihydrogen phosphate, 4) 0.2M Di-Sodium hydrogen phosphate, 5) 0.2M Di-sodium sulphate , 6) 0.2M Sodium bicarbonate , 7) 0.2M Ammonium sulphate, 8) 0.2M Tri-potassium phosphate, 9) 0.2M Tri-sodium citrate, 10) 0.1M Sodium benzoate, 11) 0.1M Potassium sorbate, 12) 0.1M Sodium propionate, 13) 0.1M Sodium chlorite, 14) 0.1M Sodium Bisulphate, 15) 0.001M CuSO4.5H2O, 16) 0.001M MnCl2, 17) 0.001M Lithium acetate, 18) 0.01M Butyric acid, 19) 0.01M Hexanoic acid, 20) 0.01M Octanoic acid. Percentage of growth inhibition compared to growth of the control. Each value represents the mean of four replicates (calculations according to Yaganza et al., 2009).

pH homeostasis in acidic conditions, or by accumulating compatible solutes to combat osmotic shifts (Vaganza et al., 2008, Ko et al., 1994; Wood et al., 2001). However, the ability of cells to accumulate the compatible solutes required is reduced in acidic conditions (Farwick et al., 1995; Ogahara et al., 1995).

However as construed from Fig.-1, only sodium carbonate, tri-potassiun phosphate (group-IV) followed these hypothesis where both the high osmolarity and the optimum pH difference brought about the complete inhibition in both Pcc strains whereas all other salts varied with respect to inhibition patterns which suggested that either Pcc cells are able to maintain homeostasis in conditions of either high ∆pH or high osmolarity alone by some unknown mechanisms. These results were also in contrast to the hypothesis proposed by Mellfont et al., 2003, that the decrease in the growth of bacteria with increasing osmolarity may be attributed to reduction of 'adaptation rate' and/or increase in the work needed (e.g., biosynthetic or homeostatic demands) to adapt to osmotically adverse conditions (Robinson et al., 1998) as no linearity in growth inhibition were observed with the increasing osmolarity.

3.2. Analysis of survival state of Pcc after treatment with different antimicrobial compounds.

To supplement methods for controlling bacterial soft rot of vegetables and fruits, there is a need for an antibacterial compound which is acceptable for use on the edible crops and vegetables after post harvest conditions. Previous work has shown that formulations of dichlorophen, which have been suggested for this purpose, were ineffective (Harris, 1979; Lund& Wyatt, 1979) and sodium hypochlorite gave only limited control. The purpose of this work was to evaluate the efficacy of other antimicrobial compounds which might be effective as preservative or microbial disease control agent for vegetables and fruits during or after post harvest.

3.2.1. Determination of minimum inhibitory concentration of various sanitizing agents for Pcc.

Minimum inhibitory concentration (MIC) of different compounds was determined for growth inhibition of both Pcc strains. There was no difference observed in MIC values of individual compound for both strains.

The MIC 0.6mM of copper sulphate for growth inhibition of both Pcc strains were found to be lower than reported earlier for fire blight causing Erwinia amylovora, which was 2 mM (Mills et al., 2006). The MIC of acetic acid, sodium hypochlorite, hydrogen peroxide and peracetic acid were found 0.4%, 200ppm, 5mM and 0.15% respectively, which were in the range generally used for sanitization of fresh fruits and vegetables as described by US-FDA. The MIC of sodium propionate, potassium sorbate and sodium benzoate were 20mM, 80mM, 10mM respectively for both Pcc strains. These three organic salts have been reported elsewhere for their preservative properties. The sub-lethal or injury effect of these compounds was analyzed on Pcc strain BR1 based on these MIC values.

3.2.2. Bactericidal and injury effects of different antimicrobial compounds on Pcc.

Many of the sub-lethal stresses (i.e., acid, salt, preservatives etc.) commonly used to control bacterial growth and/or survival in foods is known to induce injury to micro-organisms (Wong and Wang, 2004). An injured cell can be defined as an increased sensitivity of components to growth media that are not normally Inhibitory (Ray et al., 1989; Mackey, 2000; Bogosian and Bouneuf, 2001). Injured cells could be detected and enumerated based on their differential abilities to form colonies on nonselective medium such as Brain heart infusion agar (BHIA) but not on selective agar media such as Violet Red Bile Agar (VRBA) (Wu, 2008). Injury rate determination was dependent on the type of selective medium used and also on the physiological stage of bacteria tested (Beuchat and Lechowich, 1968). Bactericidal and injury effect of acetic acid on Pcc strain BR1.

Injury effect of different concentration of acetic acid (AA) on Pcc strain BR1 ranging from 0.0-0.6% was checked for a time period of 6 min or treated with 0.3% AA for different periods of time (0 to 10 min). As shown in Fig.-2, correlation was observed between injury rate and concentration of AA to cause killing effect on Pcc strain BR1. Upon exposure to 0.3% AA for 6 min, 99.7% (or 2 log units) of Pcc cells of both strains were killed. The number of viable Pcc BR1 cells as determined on BHIA was reduced from 5.4 Ã- 109 to 2.7 Ã- 106 CFU/ml following acid treatments. Among surviving of Pcc BR1 cells, only a small fraction (1.0 Ã- 103 CFU/ml) was able to form colonies on VRBA, indicates 99.99% (or more than 3 log units) of surviving cells were injured (Fig.-2A).

A positive correlation was observed between the exposure time and killing or injuring effects (Fig.-2B). The longer the exposure time, the higher death rates were observed. With both Pcc strains, following the exposure to 0.3% AA for 6 min, more than 97% of cells were killed and more than 99.9% (or 3 log units) of the surviving cells were injured. Data showed that exposed cells to low concentrations of AA can cause death and injury in Pcc strain BR1. Those cells capable of forming colonies on non-selective agar but not on selective agars were considered "injured" as previously defined (Busta, 1976; Ray, 1979). Injury rate determination was dependent on the type of selective medium used and also on the physiological stage of bacteria tested (Beuchat and Lechowich, 1968). Nevertheless, an increasing number of reports have shown that washing fresh produce and sprouting seed with acetic acid (AA) was effective for suppressing fungal decay (Delaquis et al., 1999), Escherichia coli O157:H7 (Wisniewsky et al., 2000), Salmonella typhimurium (Dickson, 1992), and Yersinia enterocolitica (Karapinar and Gönül, 1992) on fresh produce or meat products. AA is generally recognized as safe (GRAS) and has been approved by the United States Food and Drug Administration for use as a food additive or as a disinfectant for animal carcasses (FDA, 1982). Bactericidal and injury effect of peracetic acid on Pcc BR1.

Peracetic acid (PAA) was used as a modified sanitizer being composed of hydrogen peroxide and acetic acid. Survival and injury effect of peracetic acid against Pcc BR1 was studied with a concentration range varying from 0.0-1.25% of PAA for 4 minutes or treated with 0.25% PAA for different periods of time (0 to 10 min). Upon exposure to 0.25% PAA for 4 min, 99.9% (3 log units) of Pcc BR1 cells were killed. The number of viable EccBR1 cells as determined on BHIA was reduced from 3.0 Ã- 109 to 4.0 Ã- 107 CFU/ml (2 log unit reduction) followed by acid treatments (Fig.- 2C).

Log injury in both strains of Pcc was in positive correlation as increase and remain almost constant with the increasing concentration and finally decrease at very high concentration

Fig.-2 Antimicrobial action of [A, B] acetic acid and [C, D] peracetic acid on Pcc BR1 as affected by acid concentration and exposure time.

(>1.5) as the survival of population decreased. In time course exposure of 0.25% PAA, log injury of Pcc BR1 cells was increase drastically at 6 minutes (Fig.-2D). Peracetic acid's primary use in food processing and handling is as a sanitizer for food contact surfaces and as a disinfectant for fruits, vegetables, meat, and eggs (Evans, 2000). PAA can also be used to disinfect recirculated flume water (Lokkesmoe and Olson, 1993). Other uses of PAA include removing deposits, suppressing odor, and stripping biofilms from food contact surfaces (Block, 1991; Mosteller and Bishop. 1993; Fatemi and Frank 1999). PAA also inactivates enzymes that are responsible for discoloration and degradation, such as peroxidase in the browning of potatoes (Greenspan and Margulies, 1950). Although the antimicrobial effect in vitro of O2 - and H2O2 against phytopathogenic bacteria and fungi has been reported (Doke 1987; Ouf et al. 1993). Detoxification of H2O2 is probably an important factor, since this molecule is able to diffuse across membranes and cause multiple biological effects. Indeed, phytopathogenic bacteria possess enzymes that prevent oxidative damage, such as catalases and superoxide dismutases (Katsuwon and Anderson 1989; Klotz and Hutcheson 1992). Bactericidal and injury effect of sodium hypochlorite on Pcc BR1.

Sodium hypochlorite is generally being used as a sanitizer in various food industries. It is generally used at a concentration of 100-1000 ppm. In this study a range of 0-200 ppm were taken to check the bactericidal and injury effects in Pcc cells.

As shown in Fig.-3A, log injury of cells higher in Pcc BR1 strain as increase in concentration of sodium hypochlorite above 125 ppm dose. Incubation of the Pcc cells with 100ppm sodium hypochlorite for different time interval under similar conditions showed that log injury of Pcc BR1 cells increases at 15 minutes and then remained constant thereafter till 25 minute with the decrease in the survival of cells. Survival percentage of Pcc BR1 cells were found 10% only at 10 minutes and then decreased drastically (Fig.-3B).

Hypochlorite (chlorine) wash solutions were used as a treatment of cut potato seed and widely used for surface sterilization of the seeds and plant tissues used in biochemical studies and pathogen isolation (Abdul-Baki, 1973; Abdul-Baki and Moore, 1979). Hypochlorite's use as a disinfectant in the wash water in potato packing plants (Grigg and Chase. 1967) is also important. The effectiveness of hypochlorite solutions against bacterial potato pathogens has been well documented (Letal, 1977, Lund and Wyatt, 1979). In addition to having good antimicrobial properties, seed treatments must not interfere with wound healing. Preferably, they should promote wound healing. Grigg and Chase (1967), found increasing rates of hypochlorite to 5000 ppm C1 in the wash water promoted the development of a thicker and more uniform protective layer on the cut surface of potatoes. In present studies sodium hypochlorite was found effective to cause log injury and cell death in Pcc cells. Bactericidal and injury effect of copper sulphate on Pcc BR1.

A positive correlation was observed between the killing or injury rate and the concentration of copper sulphate tested (Fig.-3C). A survival percentage decreased slowly to 9.13% at 200µM concentration at 10 min exposure. Log injury was increased exponentially with the increasing concentration of copper sulphate ranging from 50-300 µM on incubation of 10 min of Pcc BR1 cells, whereas survival cells percentage was started from around 60% reduced subsequently with increase in concentration under similar conditions. Upon exposure to 300μM for 10 min, 99.5% (or more than 2 log units) of Pcc BR1 cells were killed. Among surviving of Pcc BR1 cells at 200 µM, only a small fraction (1.0 Ã- 102 CFU/ml) was able to form colonies on VRBA, indicating that 99.99% (or more than 2 log units) of surviving cells were injured. Parallel to that log injury raised as higher as around 2.5 in Pcc BR1. Significant results were obtained when Pcc BR1 was shown to have log injury as high as 2.5 at 15 min, even on prolonged incubation with 150µM CuSO4 up to 25 minutes with sufficient high log injury was observed in Pcc BR1 (Fig.-3D).

Fig.-3 Antimicrobial action of [A,B] sodium hypochlorite and [C,D] copper sulphate on Pcc BR1 as affected by copper concentration and exposure time.

Copper compounds have been reported to be effective against several plant pathogenic bacteria including Erwinia spp. (Blom and Brown 1999; Coto and Wang 1995), Pseudomonas syringae pv. syringae (Sheck and Pscheidt 1998), and Xanthomonas campestris (Jones et al. 1991; McGuire 1988; Marco and Stall 1983). However, alternative measures of control need to be explored since the development of resistance to copper has already been documented in this and other Erwinia species (Kyeremeh et al. 1998; Loper et al. 1991). This study shows that the copper sulphate cause injury effect while inhibiting growth of bacterial plant pathogens can also be used to reduce populations of Ecc. Bactericidal and injury effect of preservatives on Pcc BR1

As described in above section of salts effects on growth of Pcc, different mechanisms have been suggested to explain the antimicrobial effects of the different organic and inorganic compounds, including inhibition of several steps of energy metabolism (benzoate, bicarbonate, propionate and sorbate) (Freese et al., 1973; Wedzicha, 1984; Kabara and Eklund, 1995).

Many weak acids such as propionic acid, benzoic acid and sorbic acid were typically used as preservatives in food industry to prevent many pathogens from food spoilage (Cunningham et al., 2009). A range of 0-20 mM was used for sodium propionate and sodium benzoate for analysis of injury and bactericidal effect on Pcc BR1 strain by incubation for 4 and 5 minutes respectively. As shown in Fig.- 4A, the log injury of Ecc BR1 cells increased with the increasing concentration of sodium benzoate while the survival percentage decreased and approached 0.1 % at 20 mM concentration. Following the prolonged exposure of 12.5mM sodium benzoate, the higher death rate was observed (Fig.-4B). Potassium sorbate was used in a range of 10-120mM concentration with 20 minutes exposure time. It was observed that the log injury reached >2 at 60mM concentration with complete growth inhibition (Fig.- 4C). Above 20 minute exposure at 40mM concentration, the high death rate was observed (Fig.-4D). In case of treatment with sodium propionate at range of 2.5 to 20 mM, linear increase in log injury with respect to increase in concentration was found (Fig.-4E). The positive correlation was observed between exposure time and killing or injuring effects of sodium propionate on Pcc BR1 cells (Fig.-4F). This indicates that used preservatives controlling growth of Pcc BR1 with permissible amount and small exposure time.





Fig.-3 Antimicrobial action of [A,B] sodium benzoate; [C,D] potassium sorbate and [E, F] sodium propionate on Pcc BR1 as affected by copper concentration and exposure time.

Benzoic acid has been used in different types of acidic food products, although it is mainly used in fruit preservation. Also it is used in combination with sorbic acid for confectionary and other types of products. Benzoic acid is permited in concentrations of up to 1500 ppm in various food products (European Union, 1995). Propionic acid inhibits moulds and Bacillus spores, but not yeasts to the same extent, and has therefore been the traditional choice for bread preservation (Ponte and Tsen, 1987). Sorbic acid is considered to be more effective than propionic acid. It inhibits both moulds and yeasts, and is used in a broad variety of food products (Sofos and Busta, 1981). In most countries sorbic acid is a substance generally recognized as safe and permitted at a concentration of 1,000 to 2,000 ppm (Lund and Eklund, 2000; Somolinos et al., 2007).