Impact of Nutrients, pH, Temperature, and Water on Rhizopus & Gilbertella, Study notes of Biology

Download Impact of Nutrients, pH, Temperature, and Water on Rhizopus & Gilbertella and more Study notes Biology in PDF only on Docsity! SUMMARY Understanding the impact of environmental factors favouring natural niches of plant pathogens is a key component toward designing efficient management strategies. Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. and Gilbertella persicaria (E.D. Eddy) Hesseltine are two economically important pathogens that are commonly associated in simultaneous infections of many stone and pome fruits. The ability of R. stolonifer and G. pesicaria to assimilate diverse carbon (C) and nitrogen (N) sources and the effect of pH and the interaction tem- perature/water potential (Ψ) on their conidial germina- tion and growth were investigated. The fungi were grown on a liquid medium containing D-glucose, su- crose, fructose, maltose, or raffinose as C sources and urea, NH4NO3, NaNO2, glycine, or glutamine as N sources. The biomass production of R. stolonifer was the greatest when sucrose and fructose were used as C sources, respectively, whereas biomass production of G. persicaria was greater on fructose and glucose than on other sources. Glutamine and sodium nitrite (NaNO2) used as N sources yielded the highest biomass for R. stolonifer and G. persicaria, respectively. Sporangiospore germination and mycelial growth of R. stolonifer and G. persicaria were optimal between pH 3 and 10 and were totally inhibited at pH ≤2.5. The two fungi were affect- ed by the interaction of temperature and Ψ. Both R. stolonifer and G. persicaria were sensitive to low temper- atures (<10°C) and were completely inhibited at Ψ lev- els of -17 and -18.3, respectively. The nutrients utiliza- tion, pH, and Ψ x temperature profiles for sporan- giospore germination and mycelial growth were to some extent similar between the two species corroborating that R. stolonifer and G. persicaria occupy similar eco- logical niches. Identifying ecophysiological determi- nants of these commercially important pathogens will contribute to our understanding of their physiology and epidemiology and will aid in developing appropriate models for disease prediction. Corresponding author: A. Amiri Fax: +1.813.6340001 E-mail: achouramiri@gmail.com Key words: Ecophysiology, nutrient assimilation, en- vironment, ecological niches. INTRODUCTION Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. and Gilbertel- la persicaria (E.D. Eddy) Hesseltine are two major pre- and post-harvest pathogens of stone fruits worldwide. In addition, R. stolonifer is an important pathogen of pome fruits, berry fruits, and sweet potatoes (Splittstoesser, 1979) and frequently contaminates food products as well as animal feeds. For instance, R. stolonifer and other Rhizopus spp. are amongst the most common fungi that develop in high moisture grain such as sorghum, barley and wheat products (Christensen and Sauer, 1982; Rabie and Lüben, 1984) and are also involved in the spoilage of meat and meat products (Jay, 1979). On the other hand, G. persicaria is mainly patho- genic to plants and has a potential to cause economic loss to apricot and peach (Ogawa and English, 1991; Ginting et al., 1996), pear (Mehrota, 1964a), apple (Mehrota et al., 1971) and tomato (Mehrota, 1964b). On stone fruits, primary infections occur in the or- chards generally a few weeks prior to harvest and dur- ing storage. Inocula of the two fungi originate from or- ganic material on the orchard floor or in the soil (Ogawa et al., 1995; Ginting et al., 1996). Both fungi can infect ripe fruits of the same tree simultaneously, sporulate readily, and release large amounts of air-borne spores to contaminate healthy fruits on the same or neighboring trees. Insects feeding on sporulating fruits can serve as vectors for dissemination of Rhizopus and Gilbertella spp. to healthy fruit on the trees (Ogawa et al., 1995). It is not completely clear which pathogen is more common; however, G. persicaria has been more commonly reported than R. stolonifer in South Carolina and California (Ginting et al., 1996; Michailides et al., 1997). Distinguishing the two species can be challeng- ing as they both produce abundant black sporan- giospores, which could be easily confused with infec- tions caused by Mucor spp. Fruits are particularly susceptible to fungal spoilage because of their high content in sugars and other nutri- Journal of Plant Pathology (2011), 93 (3), 603-612 Edizioni ETS Pisa, 2011 603 EFFECT OF NUTRIENT STATUS, pH, TEMPERATURE AND WATER POTENTIAL ON GERMINATION AND GROWTH OF RHIZOPUS STOLONIFER AND GILBERTELLA PERSICARIA A. Amiri1, W. Chai2 and G. Schnabel2 1 University of Florida, Gulf Coast Research and Education Center, Wimauma, 33598 Florida, USA 2 Department of Entomology, Soils, and Plant Sciences, Clemson University, Clemson, 29634, SC, USA 007_JPP643RP(Amiri)_603 15-11-2011 17:44 Pagina 603 604 Parameters affecting growth of R. stolonifer and G. persicaria Journal of Plant Pathology (2011), 93 (3), 603-612 ents, low pH, and richness in available water. Further- more, fruits are subjected to multiple changes in soluble solid and volatile compounds, flesh firmness, and water content during their growth in the field and during stor- age. Such conditions may differentially favor fungal in- fections including those caused by R. stolonifer and G. persicaria. In most fruits, sugar metabolism is primordial for fruit development and organoleptic qualities with su- crose, glucose, and fructose being respectively the main sugars in peaches (Genard et al., 2003). While glucose and fructose are accumulated at constant rate through- out development and ripening (Souty et al., 1998), su- crose, which is the main sugar in peach, accumulates rapidly in the last few days of maturation on the tree (Layne and Bassi, 2008). The utilization of sugars as car- bon sources has been investigated in several ectomycor- rhizal fungi (Hampp et al., 1995; Martin et al., 1998; Grelet et al., 2005; Deveau et al., 2008), ascomycetous yeasts (Leandro et al., 2009), and some filamentous fun- gi such Aspergillus spp. and Neurospora crassa (Forment et al., 2006; Xie et al., 2004). However, the potential im- pact of change in sugar content during fruit growth, ripening and storage on disease development is under- investigated in many fungal pathogens such as R. stolonifer and G. persicaria. Many fungal species can uti- lize low molecular-weight compounds such as mono- and disaccharides but not large molecules composed of these same small subunits. In addition, one fungal species may have the ability to assimilate one particular sugar for germination of its sporangiospores but not for mycelial growth and sporulation (Agnihotri, 1969). The determination of sugar utilization as carbon sources could help in determining niche overlap indices (Wilson and Lindow, 1994) which will determine ecological simi- larity between different fungi that occupy similar niches. Organic acid metabolism is another crucial determi- nant of fruit quality. In peaches, malate and citrate are the two major organic anions (Layne and Bassi, 2008) and account for most of the titratable acidity. Their ac- cumulation occurs mainly in early stages of fruit growth and progressively decreases during fruit ripening induc- ing consequently, a decrease in fruit acidity. The pH is a well known growth regulator in many fungi and its ef- fect on fungal growth has been extensively studied for several fungal species in vitro. For sugar metabolism, however, the impact of acidity change during fruit growth and storage on Rhizopus and Gilbertella rots development is unknown. Environmental factors such as temperature, water potential, and their interaction affect the competitive- ness and co-existence of spoilage fungi in fruits and oth- er food ecosystems. The effect of temperature on spo- rangiospore germination, sporulation, or disease devel- opment has been investigated in R. stolonifer (Miller et al., 1959; Smith and McClure, 1960; Pierson, 1965; Dennis and Blijham, 1980) but to a lesser degree in G. persicaria (Ginting et al., 1996; Michailides et al., 1997). However, there is a paucity of knowledge on the impact of water potential, interaction between temperature and water potential, and other conditions such as pH and carbon and nitrogen sources availability on competitive- ness and pathogenicity of the two aforementioned species. Water content in fresh fruit may not be a limit- ing factor for fungal growth; however the extent to which these pathogens can survive and assimilate sugars and other nutrients from the soil, cankers, or mummies on which they overwinter may determine their ability to germinate, sporulate, ensure the continuity of their de- velopmental cycles, and will define their ecological nich- es. Control of Rhizopus rot relies mainly on prophylac- tic measures to reduce inoculums and pre- and posthar- vest fungicide applications (Northover and Zhou, 2002), whereas no efficient chemical control strategies currently exist for Gilbertella rot management. Hence, understanding the development of these species on dif- ferent substrates and under different environmental conditions may constitute a basis for developing effi- cient and sustainable disease methods for their control. The objectives of this study were to evaluate the ability of R. stolonifer and G. persicaria to utilize a range of car- bon and nitrogen sources and to investigate the effect of pH, water potential, temperature, and their interaction on sporangiospore germination and mycelial growth. MATERIALS AND METHODS Origin, maintenance, and inoculum production from fungal isolates. Two R. stolonifer isolates (Rh.by2 and Rh.by5) and two G. persicaria isolates (Gp.pch1 and Gp.my1) were used in this study. G. persicaria isolates were obtained from decaying peach fruit collected from two commercial orchards in Pickens and Edgefield counties, South Carolina (USA), while the R. stolonifer isolates were obtained from the USDA Research Sta- tion, Byron, Georgia. The isolates were characterized at a molecular level based on the internal transcribed spac- er ITS1 and ITS4 regions (White et al., 1990). Isolates were stored at -80°C on silica gel (grade 40; Sigma- Aldrich, USA) and new cultures were grown from this stock on potato dextrose agar (PDA) medium for each experiment. Sporangiospores from R. stolonifer and G. persicaria were obtained from 4-day-old PDA cultures. Sporangiospores from PDA cultures were washed in 15 ml of sterile distilled water containing 0.05% Tween 80, filtered through cheesecloth and the concentration of the suspension was determined using a haemocytometer. Media. The basal medium (BM) used to assess the uti- lization of carbon and nitrogen sources contained (g l-1): K2HPO4 (1.0); MgSO4.7H2O (0.2); NaCl (0.07); ZnSO4.7H2O (0.04); ferric EDTA (0.01) (Grelet et al., 007_JPP643RP(Amiri)_603 15-11-2011 17:44 Pagina 604 Journal of Plant Pathology (2011), 93 (3), 603-612 Amiri et al. 607 growth was drastically reduced at Ψ level of -9.8 MPa and inhibited at lower Ψ levels. The mycelial growth of both fungi tested was affected by the nature of solute added to PDA to generate different Ψ levels. Based on GLM analysis, the growth was significantly reduced with NaCl compared to glucose at Ψ levels ≤ -9 MPa (Fig. 4). Effect of water potential and temperature interac- tion on germination and mycelial growth. Overall, spo- rangiospore germination of R. stolonifer and G. persi- caria was inhibited in a temperature range of 5 to 10°C regardless of the Ψ level (Fig. 5). Both species germinat- ed at a 100% rate at Ψ levels up to -6 MPa at 20°C. At this temperature, germination of stolonifer and G. persi- caria was reduced to 53 and 39%, respectively, at -14 MPa. Germination of G. persicaria decreased sharply to 35% at 30°C and -9.8 MPa, whereas both species where completely inhibited at -14 MPa. Neither R. stolonifer nor G. persicaria grew at tem- peratures below 10°C regardless of the Ψ level (Fig. 5). Optimal growth for both species was observed at 20°C and Ψ levels up to -6 MPa. Mycelial growth of both species decreased at 30°C for all Ψ ranges and was com- pletely inhibited by a -14 MPa/30°C combination. DISCUSSION Fungi have a variety of requirements for organic en- ergy, space, water, other nutrients and oxygen for growth and reproduction. It is primordial to consider that fungal contamination in plants and foods is not a result of one single, but communities of genera and species that compete for nutrients and other require- Fig. 2. Effect of water potential on sporangiospore germina- tion of Rhizopus stolonifer and Gilbertella persicaria after 48 h at 20°C. Data are the mean of 18 replicates from two isolates per species across two experimental runs. Vertical bars indi- cate the standard deviations. Fig. 3. Temporal effects of water potential on mycelial growth of Rhizopus stolonifer and Gilbertella persicaria at 20°C. Data are the mean of 18 replicates across two experimental runs. Vertical bars indicate the standard deviations. Fig. 4. Mycelial growth of Rhizopus stolonifer and Gilbertella persicaria on potato dextrose agar with different Ø values ob- tained with sodium chloride (solid line) and glucose (dashed lines) at 20°C. 007_JPP643RP(Amiri)_603 15-11-2011 17:44 Pagina 607 ments. To ensure the continuity of its developmental cy- cle, a fungus must reduce the effect of potential com- petitors or utilize effective competitive mechanisms. Although R. stolonifer has a wider host range than G. persicaria, these two pathogens are commonly observed or recovered at similar proportions from infected or symptomless stone fruits (Ginting et al., 1996; Michailides et al., 1997). The present study is the first attempt to evaluate and compare the ability of these species to utilize different C and N sources and to grow under different environmental conditions. R. stolonifer and G. persicaria were able to grow on a basal medium (BM) with only a C (glucose) or an N (glutamine) source. Nevertheless, all C and N sources supplemented into the BM had a significant positive effect on biomass production. Out of five C sources tested, R. stolonifer grew best on two important sugars of fruits, i.e. the dis- accharide sucrose composed of two units of glucose and fructose and the monosaccharide fructose. Overall, bio- mass production of G. persicaria was slighter compared to R. stolonifer, and was preferentially higher on mono- saccharides. This may denote a wider ecological niche for R. stolonifer over G. persicaria, although parameters other than nutrient utilization may define competitive- ness. Our results also show a pattern in sugar utilization that may help in understanding the pathogenicity of these species on stone fruits. In fact, R. stolonifer used better sucrose over fructose and glucose contrary to G. persicaria. Sucrose, fructose and glucose in proportion of about 3:1:1 are the mains sugars in peaches (Genard et al., 2003; Vizzoto et al., 2002). Glucose and fructose accumulate throughout fruit development but their ra- tio decreases during maturation (Layne and Bassi, 2008), whereas sucrose accumulates at a higher rate (>50% of total dry weight) during the last few days of maturation on the tree (Souty et al., 1998; Bregoli et al., 2002; Vizzoto et al., 2002). It is therefore plausible to hypothesize that G. persicaria may be more pathogenic on fruit at early stages of development whereas R. stolonifer infects better ripen fruit richer in sucrose. This may also explain the fact that G. persicaria is more frequently encountered than R. stolonifer on fruits sam- pled from orchards in South Carolina and California (Ginting et al., 1996; Michailides et al., 1997). Com- pared to the other sugars tested, the trisaccharide raffi- nose was the least used sugar by G. persicaria and more 608 Parameters affecting growth of R. stolonifer and G. persicaria Journal of Plant Pathology (2011), 93 (3), 603-612 Fig. 5. Effect of water potential and temperature on sporangiospore germination (after 48 h) and mycelial growth (after 16 days) of Rhizopus stolonifer and Gilbertella persicaria. Data are the mean of 18 replicates across two experimental runs. Vertical bars are the standard deviations. 007_JPP643RP(Amiri)_603 15-11-2011 17:44 Pagina 608 particularly by R. stolonifer. This molecule is composed by galactose, fructose, and glucose and is found in many vegetables and fruits. Its weak utilization might be relat- ed to the fact that the two fungi lack α-galactosidase ac- tivity that can hydrolyze raffinose to D-galactose and su- crose. Based on their ability to assimilate different N sources, fungi have been divided into two classes: those able to assimilate molecular nitrogen (N2) and those which grow preferably on inorganic and organic nitro- gen sources (nitrate, nitrite, or ammonium and organic N compounds) (Lilly, 1965). While N2 utilization has not been clearly shown in fungi, the majority of fungi can efficiently grow on substrates supplied with either an inorganic or an organic N source. Based on our re- sults, R. stolonifer and G. persicaria belong to the sec- ond class of fungi being able to assimilate all N sources, although R. stolonifer showed a clear preference for or- ganic N compounds. Both fungi produced maximum biomass on glutamine (glutamic acid), which was re- ported to be a suitable N source for many microorgan- isms such as Alternaria tenuis, Aspergillus niger, and Tri- choderma spp. (Singh and Tandon, 1970; Apsmo et al., 2005; Rossi-Rodrigues et al., 2009). A likely explanation for this preference may be related to the primordial role of glutamine in the nitrogen utilization cycle in fungi (Garraway and Evans, 1984). Seabi et al. (1999) found that R. stolonifer can grow adequately on glutamatic acid but not on ammonia (NH4Cl), nitrate (KNO3), or nitrite (NaNO2) amended to 0.1 g l -1 in a solid medium. The same authors, however, found that other Rhizopus species such as R. oryzae were able to grow on NH4Cl and KNO3 but not on NaNO2. In our study, N sources were added at a final concen- tration of 10 g l-1 to a liquid medium and our findings are more consistent with those of Inui et al. (1965) who reported that Rhizopus species were unable to utilize 21 g l-1 nitrate in a liquid medium as well. Although both fungi were able to germinate and grow at pH value up to 10 in this study, it has been reported that nitrite com- pounds may cause an increase of pH in the medium and inhibit the growth of some mycetes (Agnihotri, 1968). Furthermore, ammonium (NH4+), a relatively poor N source for the fungi tested, has been linked to the degradation of DNA and proteins in fungi (Pinon, 1977; Goller et al., 1998). To our best knowledge, there are no reports in the literature on the ability of G. persi- caria to use C or N sources, however in this study, G. persicaria used more efficiently different N sources in- cluding nitrite than R. stolonifer. This capacity may fa- vor the overwintering of G. persicaria over R. stolonifer in soils and mummified fruits and would place G. persi- caria among the succession of microorganisms more suitable for nitrogen mineralization of organic matter. Fungi generally are known to grow well at acidic con- ditions similar to those found in fruit at commercial ma- turity. Environmental H+ concentration (pH) is thought to have little direct effect on fungal metabolism due to the buffering system in hyphae and conidia (Dix and Webster, 1995). However, such conditions may influ- ence their enzymatic activity and therefore their ability to colonize and infect their hosts (Enokibara et al., 1993; Aleandri et al., 2007). In consistence with previ- ous reports, R. stolonifer and G. persicaria were only in- hibited in very acidic conditions (pH ≤ 2.5) but not af- fected at alkaline pH although mycelial growth was re- duced at pH higher than 9. For example, zygospore production by G. persicaria on PDA medium decreased at pH below 4 but not at pH values up to 10 (Michailides et al., 1997). Mycelial growth of R. stolonifer was optimal at pH 7 and was reduced at 4≤ pH ≥ 9 in a liquid medium (Adisa, 1983). Under normal condition (temperature, water availability), it is unikely that pH will be a limiting factor for the growth or spo- rangiospore germination of these species. However, during the growth of stone fruits, pH can decrease sometimes below 3.25 before harvest due to a high con- tent in malate and citrate anions, especially in high-acid cultivars (Kakiuchi et al., 1981; Moing et al., 1998). While the effect of temperature on growth of R. stolonifer has been studied extensively, there is paucity in studies regarding G. persicaria and the effect of water potential and temperature interaction on both fungi. R. stolonifer and G. persicaria were sensitive to low temper- atures since no germination or mycelial growth was ob- served below 10°C regardless of the Ψ level, whereas optimum germination and growth were observed be- tween 15 and 30°C at Ψ levels up to -6 MPa. Previous studies reported a temperature range between 5 and 25°C for optimal germination and growth of R. stolonifer (Miller et al., 1959; Pierson, 1965; Adisa, 1983). Zygospore production by G. persicaria was inhib- ited at temperatures ≤15°C and significantly reduced at 25°C compared to 20°C (Michailides et al., 1997) and symptoms of G. persicaria developed on peaches incu- bated at 21-24°C (Ginting et al., 1996). Our results and other reports suggest that cooling the fruit immediately after harvest to temperatures below 4°C is one way to reduce postharvest disease infections due to these two pathogens. However, once the fruit are exposed again to room temperature, the risk might be elevated since these species have been show to keep their viability after long period of chilling (Smith and McClure, 1960; De- nis and Blijham, 1980). The minimum Ψ observed for germination for R. stolonifer and G. persicaria was -14 MPa at 20°C. Other studies have reported that R. stolonifer has germinated at Ψ values as low as -8 to -10 MPa (Homer and Anag- nostopolous, 1973) and -26 MPa (0.84 aw) (Hocking and Miscamble, 1995) at 25°C. The minimum Ψ ob- served for mycelial growth was -9.8 MPa, lower com- pared to germination which is in consistence with many Journal of Plant Pathology (2011), 93 (3), 603-612 Amiri et al. 609 007_JPP643RP(Amiri)_603 15-11-2011 17:44 Pagina 609