Abstract. Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores.
J. Phytopathology 118, 3—8 (1987) © 1987 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0031-9481
Horticultural Crops Quality Laboratory, USDA-ARS, Beltsville, Md., U.S.A. and Botany Department, University of Maryland, College Park, Md., U.S.A.
Influence of Temperature and Relative Humidity on Germinability of Mucor piriformis Spores REGINA B . BERND, G . A . BEAN, W . S. CONWAY and H. E. MOLINE Authors' addresses: REGINA B. BERND, National Center for Genetic Resources and Biotechnology, CENARGEN/EMBRAPA, P.O. Box 10.2372, 70.77D-Brasilia, DF (Brazil). G. A. BEAN, Department of Botany, University of Maryland, College Pk., MD 20742 (U.S.A.). W. S. CONWAY and H. E. MOLINE, United States Dept. of Agriculture, Agricultural Research Service, Horticultural Crops Quality Laboratory, BARC-W, Beltsville, MD 20705 (U.S.A.). Hth one figure Received November 5, 1985; accepted March 17, 1986
Abstract Studies were conducted to determine the influence of temperature and relative humidity (RH) on germinability and viability of Mucor piriformis spores. Spores did not survive when stored at 35 °C and their survival rate decreased rapidly at 30 °C; however, spores remained viable for more than 1 year at 0°C. RH also significantly affected spore viability. Spores held at 26 °C and 100% RH no longer germinated after 35 days, while those held at 75 or 90% RH germinated for 65 days. At 20 °C, RH had little effect on spore germinability. The effect of temperature and RH on percentage spore germination also varied. At all temperatures studied, spore viability decreased more rapidly with time at 100% RH than at 75 or 90% RH. The least favorable temperature-humidity combination, 30 °C and 100% RH, decreased spore germination from 100% to less than 1 % in 14 days.
Zusammenfassung Einflufi von Temperatur und relativer Luftfeuchtigkeit auf die Keimfahigkeit von Mucor piriformis Sporen Es wurden Untersuchungen durchgefiihrt, um den Einflufi von Temperatur und relativer Luftfeuchtigkeit (RH) auf die Keim- und Lebensfahigkeit von Mucor piriformis Sporen festzustellen. Die Sporen Iiberlebten eine Lagerung bei 35 °C nicht, und ihre Uberlebensrate verringerte sich sehr schnell bei 30 °C. Jedoch bei 0°C blieben die Sporen uber ein Jahr lebensfahig. Die Lebensfahigkeit der Sporen wurde auch durch die RH signifikant beeinflufit. Sporen, die bei 26 °C und 100% RH gelagert wurden, keimten nach 35 Tagen nicht mehr, wahrend Sporen, die bei 75 oder 95% RH gelagert U.S. Copyright Clearance Center Code Statement: 0031-9481/87/1 801-0003$02.50/0
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wurden, 65 Tage lang keimten. Bei 20 °C hatte die RH kaum Einflufi auf die Sporenkeimfahigkeit. Die Auswirkung von Temperatur und RH auf die prozentuale Sporenkeimung war auch unterschiedlich. Bei alien untersuchten Temperaturen verringerte sich die Sporenlebensfahigkeit schneller bei 100% RH als bei 75 oder 90 % RH. Bei der ungiinstigsten Temperatur-Liiftfeuchtigkeitskombination — 30 °C und 100% RH — verringerte sich die Sporenkeimung von 100% zu weniger als 1 % innerhalb 14 Tagen.
The importance of post-harvest diseases is illustrated by the heavy losses that occur between harvest and consumption of fruits and vegetables. A major cause of these losses is attributed to decomposition by microorganisms (ECKERT and SoMMER 1967, RYALL 1965). Mucor pirifonnis Fisher causes post-harvest decay of numerous fruits and vegetables, including strawberries, raspberries, blackberries, pears, apples, and stone fruits (BERTRAND and SAULI-CARTER 1980, DENNIS and MouNTFORD 1975, LoPATECKi and PETERS 1972, SMITH et al. 1979). During March 1984, M. piriformis was responsible for the loss of 5,000 bushels of Delicious apples at a single fruit packing company in Pennsylvania, representing a loss of approximately $30,000 (J. Rice, pers. comm.). Wounding of fresh fruits and vegetables during harvesting and handling cannot be completely avoided (ECKERT 1975, SoMMER 1982). Wounded produce is especially susceptible to infection by microorganisms that contaminate it in the packing house (BARKAI-GOLAN 1966, SMITH et al. 1971). Thus, inherent in the successful control of post-harvest pathogens such as M. piriformis are measures used to reduce the level of inocolum in storage facilities. Manipulation of temperature and relative humidity (RH) are the most common physical factors recommended for control of post-harvest decay (ECKERT 1977, RIPOON 1980, SPOTTS and PETERS 1982). Low temperature storage maintains the quality of many fruits and vegetables by retarding growth and sporulation and by suppressing metabolic activity of many fungi (DENNIS and BLIJHAM 1980, MATSUMOTO et al. 1969). Mucor piriformis presents a unique problem because it causes decay at 0°C and cannot be controlled by approved fungicide treatments. There are conflicting reports concerning the response of M. piriformis spores to temperature and RH (SNOW 1949, SPOTTS 1985, SPOTTS and PETERS 1982). The objective of this study was to investigate the interaction of temperature and RH on survival of M. piriformis spores under conditions similar to those which might be encountered during the harvesting, storage, and marketing of fresh produce.
Materials and Methods The strain of M. piriformis (ATCC #38314) used in this study was obtained by single spore isolation from peaches growing in Maryland. Cultures were stored in darkness at 0°C on Difco potato dextrose agar (PDA) slants. For spore production, M. piriformis was grown on PDA in darkness at 20°C for 5 days in disposable Petri dishes (100 x 15 mm). Fungal spores were transferred from culture media to autoclaved gum tree {Nyssa syhatica Marsh) wood chips. Wood chips ( 8 X 1 5 mm) were coated with spores by bringing them into contact with the surface of sporulating M. piriformis colonies growing on PDA. The chips were then placed in sterile plastic Petri dishes (100 X 15 mm) without medium. Preliminary experiments showed that spores did not germinate on the wood chips
Influence of Temperature and Relative Humidity
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and viability of spores was similar to that on glass surfaces. The dishes containing the spore laden wood chips were placed in sealed chambers (Gaspak 100 System polycarbonate jars, 130D x 230Hmm; Fisher Scientific, Silver Spring, MD), and RH's (40%, 75%, 90%, 100%) were established according to the method of WEXLER and BROMBACHER (1952) and maintained by circulating air through various proportions of glycerin-water mixtures or distilled water (100% RH) through the closed system which diaphragm pumps. RH's were monitored at least weekly using a thermoelectric dew point hygrometer (EG & G Model 880, Waltham, MA). Temperatures of 0, 10, 20,26, 30, and 35 °C were maintained by placing the RH chambers in incubators (Precision Scientific Model 818, Chicago, IL) set at the desired temperatures. Percentage spore germination was determined at weekly intervals by removing wood chips from plates and placing them for 3 min in test tubes, each containing 1 ml sterile distilled water. After agitation, the spore suspension was pipetted from the tubes and evenly distributed over the surface of PDA plates. After an incubation period of 18 h at 20°C in darkness, 100 spores selected at random on each plate were examined under lOOx magnification and the percentage germination calculated. A spore was considered viable if a germ tube was observed. Those plates with spores not germinating were held at 20^0 and re-checked daily for at least 1 week. All experiments were replicated three times.
Results The influence of temperature and RH on viability of spores of M. piriformis is summarized in Table 1. Both temperature and RH influenced the survival of spores, although temperature had the greater effect. As the temperature of incubation was increased, the survival rate of the spores decreased. Spores incubated at 0°C germinated more than 1 year later, while those incubated at 35 °C did not germinate after 24 h. At each temperature between 10 and 30 °C spores held at the lowest humidity (40%) survived for the longest time. If we examine the percentage spore germination (Fig. 1) we can see that the rate of germination dropped most rapidly at 100% RH. Although a few spores continued to germinate after 35 days at 20°C and after 14 days at 26 and 30°C, the spore survival rate was significantly reduced at 100 % RH at these three temperatures compared with 75 and 90% RH. The few spores that remained viable continued to germinate for up to 77 days at 20 °C, for 35 days at 26°C, and for 28 days at 30°C (Table 1). Table 1 Effects of temperature and relative humidity on the germinability of Mucor piriformis spores % Relative humidity 40 75 90 100
0 365 365 365 365
+ 3}'' -1- a -t- a -f- a
10 154 a 119 b 112 b 105 b
Incubation temperature (°C) 20 26 98 a 77 b 77 b 77 b
84 a 63 b 63 b 35 c
30
35
49 a 35 b 35 b 28 b
— — —
Mean number of days after which no spores germinated; spores stored at 0°C continued to germinate at termination of experiment at 1 year and those stored at 35°C did not germinate after 24 h. Numbers followed by the same letter within each column did not differ significantly (P = 0.05) according to the LSD test.
M
21
28
3S
42
Tim* »t Cxpeaura Cdays) 108-
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1 u
a.
14
2t
26
49
35
Tim* of Expocur* (days)
0 o
a. 20-
42 Tint* of
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Fig. 1. The influence of temperature and relative humidity on percentage spore germination of Mucor piriformis at various time intervals. Temperatures include A) 20 °C, B) 26 °C, and C) 30 °C. Relative humidities at each of the temperatures are 75% (•), 90% (A), and 100% (•)
Influence of Temperature and Relative Humidity
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Discussion Temperature and RH influence both the percentage germination of M. piriformis spores and the long term viability. A combination of factors may be responsible for the rapid loss in viability of M. piriformis spores when exposed to high temperature and RH. During germination, spores are more sensitive to extreme temperatures and lose viability and virulence, especially during prolonged periods of exposure to high or low temperatures (DENNIS and BLIJHAM 1980, SMITH et al. 1979). At a high RH, spore germination is stimulated (PAGEet al. 1947); however, the germinating spores are more susceptible to high temperatures and the metabolic steps involved in spore germination may be retarded or totally inhibited. Previous studies (ECKERT and SOMMER 1967) have shown that enzymes are more readily inactivated when hydrated, slowing down or inhibiting metabolic processes which occur during spore germination. High RH's stimulate spore germination; however, high temperatures may also be inhibitory to enzymatic processes that are occurring in spores causing germination to cease. Free water inside spores is normally in equilibrium with water surrounding the spore. Thus, spores held at a high RH would have a greater proportion of water in the free state and less food reserves available in the protoplasm. Therefore, the free water inside the spores, coupled with a high temperature, increases the metabolic rates and reduces food reserves in the spores for germination which could result in a rapid loss in viability of the spores (MEREK and FERGUS 1954). Two physical properties may also contribute to the lethal effect of the 30 °C and 100% RH treatment. Heat is transferred more readily in wet air (ECKERT and SOMMER 1967), and high relative humidity enables a higher temperature to be reached in spores in a shorter period of time (SMITH and WORTHINGTON 1965). Mucor piriformis spores fail to germinate at temperatures above 27 °C (SMITH et al. 1979); however, they can survive at temperatures of 30°C (Table 1). Increasing the RH drastically reduces spore survival at higher temperatures. Since the pathogen cannot be effectively eliminated with approved fungicides, a combination of high temperature and high humidity may place enough stress on the pathogen to reduce its potential pathogenicity. Sanitary practices such as steam cleaning or solarization of storage facilities and field boxes should remove residual inoculum that might cause decay of injured fruit. By understanding how environmental conditions affect spore survival and manipulating the environment to our maximum benefit we may place enough stress on the fungus to minimize its impact as a post-harvest pathogen. This work was part of a thesis submitted by R. B. BERNfD to the Botany Department, University of Maryland, College Park, MD for the M.S. degree. Use of a company or product name by the U.S. Department of Agriculture does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.
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