Penicillium In The Local Pear Supply Chain Biology Essay


The pear fruit, Pyrus communis, is classified in the Rosaceae family Mitcham et al, 2009. Pears are highly perishable fruits especially in the post-harvest phase (Mari, 2003). Pears are very sensitive to temperature changes and should be harvested at exactly the right time (Mitcham et al, 2009). In order to determine the quality of pear commodity there are several factors which assessors use (Mitcham et al, 2009; Janick, 1992) such as size, colour, flavour, aroma and texture. External factors strongly influence the consumer's initial reaction towards the fruit but the final judgment should rather be after it has been tasted (Janick, 1992).

In South Africa the pear industry, which has an allocated 12 912 hectares, is extremely important in terms of economy and export (Human, 2005) being the third largest industry in South Africa. Ceres is the largest pear producing area in South Africa with 54% growth area dedicated to pear production alone (Human, 2005). The three main pear varieties currently produced in South Africa is Packham's Triumph, Bon Chretien and Forelle having 28%, 25.1% and 15.8% dedicated hectares respectively (Human, 2005). These pear varieties are also exported in extremely large quantities with Packham's Triumph being the leader with 53.2% of the annual harvested produce exported (Human, 2005).

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Bon Chretien pears are the most susceptible pear variety prone to premature ripening making it the perfect conditions for postharvest infections to occur (de Vries, 2003). According to Sanderson and Spotts (1995) it was determined that P. expansum and P. solitum were the most abundantly isolated Penicillium species in pack houses, dump tank water system and markets. Blue mould can be caused by several Penicillium species but the most common pathogens are P. expansum, P. solitum and P. commune (Kupferman, 1993).

Penicillium species are characterized under the Deuteromycete fungal kingdom. Penicillium species are the most abundantly found microorganisms and usually the most destructive postharvest pathogens on fresh produce (Skim, 2002; Coursey and Booth, 1972). Penicillium infections can occur in the field but 90% of postharvest infectious can be initiated under environmental conditions favourable to the pathogen (Pitt, 2009). Infections can be caused while in transit, storage and in the market due to several influential factors such as temperature and moisture content (Kelman, 1989). Penicillium species are known to cause severe postharvest losses in apples and pears globally. It has also been showed that Penicillium species have increased spore levels as the packing season progresses (Sholberg, 2004). Although previous studies done by Dr. Lennox (2003) show that inoculum levels play an important role in initiating these infections and that sanitation of the pack house is of great importance prior to the fruit entering the pack line. Penicillium expansum is also very abundantly found in bins, water systems, and dump tanks and very close to the packing line which can explain the very high correlation found by Sholberg (2004) between fruit contamination and blue mould infections.

Annual postharvest fruit decay losses in fresh produce and are estimated to be 10-30% of harvested fresh produce in developing countries more so than others (Sholberg and Conway, 2004; Kelman, 1989) despite several modern technology that are used. Blue and grey mold caused by Penicillium expansum and Botrytis cinerea, respectively cause serious problem in the pear industry (Janisiewicz, 2006; Mari, 2003; Sanderson and Spotts, 1995) and cause devastating losses which ultimately have an economical impact on export commodities (Coursey and Booth, 1972). Study done by Sanderson, 1995 indicated that Penicillium species was isolated in high density and diversity. Although other postharvest pathogens were also isolated from the collection sites Penicillium species, with special reference to Penicillium expansum, was found to be the most virulent and most destructive pathogen when it came into contact with wounds (Sanderson, 1995).


Postharvest research has been neglected for a considerable amount of time due to the lack of consideration and funding (Goletti and Wolff, 1999). Postharvest research is a multidisciplinary field of study because of the diverse activities that needs to be considered when it is studies such as harvesting activities, cultural farm practices, product quality, transport and utilization (Goletti and Wolff, 1999). Postharvest research and innovations are of great importance in order to maintain quality of the horticultural crop and its products. If postharvest losses are reduced it can be beneficial to the expanding human population due to availability of microbiologically safe foods as well as beneficial to the environment because less growing areas are required and natural resources will be conserved. Postharvest research contributes towards sustainability of resources by finding alternative management strategies which accumulates in natural environment (Kader, 2003; Goletti and Wolff, 1999).

Hypothesis statement

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The inoculum load and Penicillium species diversity will change over time from the start of the growing season to the end of the season.

The inoculum load and species diversity within pear packhouse and storage areas may have an effect on the disease severity.

If Penicillium species are isolated abundantly from the atmosphere and pack house surfaces it could be anticipated that postharvest diseases can be caused by Penicillium species and this can play a role in pear quality. It is also expected that P. expansum, P. solitum and P. commune, the postharvest pear pathogens according to literature, will be isolated.


The aim of the project will ultimately be to determine the specific Penicillium populations present in the pear pack house, transport and storage environment. Penicillium species inoculum build-up will be determined over time. The inoculum levels of Penicillium spp will also be looked at to see whether there is a correlation between the inoculum load and disease incidence and severity. We will attempt to identify inoculum sources possibly contributing to fruit cross-contamination and postharvest fruit decay.


The primary objectives of the research are to:

Identify the problems associated with postharvest diseases experienced by the pear industry relating to Penicillium postharvest diseases.

Determine which Penicillium species occur most frequently

Identify inoculum sources possibly contributing to the postharvest decay

Observe population dynamics over a season in the pack house, transport vessel and on the local market

Experimental approach

Air sampling

Active and passive air sampling will be done on the farms, in the pack house, in trucks and at the local fresh produce market. In the active air sampling an automated air sampler will be used at a specific sampling site for sample collection. A sterile malt extract agar (MEA) contact plate is place on the inside of the sampler and air inflow will be performed for one minute in coldrooms where approximately 100L of air will be sampled as well as in the pack house where air will be samples for 30 seconds. The processing of the samples, through dilutions series and purification of the cultures, will be done at the University of Pretoria's Plant pathology laboratories.

Swab sampling

Specific surface areas (5cm x 5cm) in the pack house, transport vessel and storage facility will be pre-determined and are selected and swabbed in vertical and horizontal directions. Swabs are placed in the plastic storage tube which will be processed within two week. It will be kept in a cold room. Specific sample codes are used for each individual swab taken.

Processing Swab Samples

The swab samples taken are processed using the dilution series method. The swab is cut with sterile pliers into a test tube containing 9mL Ringer's solution. A dilution series is performed until the desired dilution 10-5. There will be five tubes labelled 10-1 to 10-5 with their respective 10-2 to 10-6 labelled MEA growth media plates. MEA growth media is used for the isolation, cultivation and counting of moulds and yeast due to the optimum pH levels being 5.4 or 3.5 (Harrigan, 1998). Each dilution indicates that the sample has been diluted 1000 times from its original concentration. Each swab will have replicates of three. The 100µl plated and spread with a hockey stick onto the growth medium will be incubated at 25°C for 4-6 days. Corresponding sample codes will be written down on the growth medium.

Penicillium isolations and purification

After the incubated time period, the plates containing Penicillium isolates are used to make pure cultures. A few spores of a Penicillium colony is removed with an inoculation needle and inoculated onto MEA media where it will be incubated at 25°C for 5-7 days in order to obtain pure cultures.

Grouping of isolates

All the isolates are grouped into clearly numbered groups resembling their culture characteristics according to morphological similarities. From each group a representative plate will be used for further investigation.


The representative isolates are preserved in the following manner: Subculturing of agar blocks in water and cryopreservation. Subculturing is an inexpensive and simple storage method where agar blocks are cut from the actively growing edges in 5mm x 5mm of a colony and placed in sterile water in a McCartney bottle, the rubber cap is screwed on and stored in a cold room which is between 1-10°C (Pitt, 2009). Cryopreservation is a method used to store a living organism at ultra low temperatures in order to allow its revival when needed.

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Morphological identification for Penicillium species are based on the use of three different types of growth media known as Czapek yeast extract agar (CYA), malt extract agar (MEA) and 25% glycerol nitrate agar (G25N) (Pitt, 2009). The plates are incubated at three different temperatures being 5°C, 25°C and 37°C for 7 days. Culture size, colour, exudates production etc. is taken into consideration for morphological identifications.

The molecular techniques that are used to identify the Penicillium species are DNA extraction using the Nucleospin II Plant DNA extraction kit (Macery-Nagel). Polymerase Chain Reaction (PCR) developed in the 1980s by Kary Mullis provides a sensitive and specific diagnostic technique where a specific region of a DNA strand (the DNA target) is amplified using three different temperatures. RFLP-PCR is a diagnostic analysis which can rapidly distinguish between different Penicillium species (Dupont et al, 1999). Molecular tools and diagnostic tests are used frequently in order to accurately characterise between different fungal species. RFLP-PCR is effective molecular techniques in order to differentiate between near-identical Penicillium species (Dupont et al, 1999).

Statistical design

Data will be collected at randomized selection sites on the farm, within the pack house, in the truck and on the market. Swab sampling and air sampling will be the methods used to sample surface areas. All experimentation will be repeated three times during the packing season. The collected data will be analysed statistically through making use if replicates throughout the experiment. The swab samples will be processed with a repetition of three per swab. Graphical visualization tools are used for comparisons between the different Penicillium species present and the densities they are isolated in.

Time frame


Pro Forma Project Budget

Expense Category

Estimated Expense

Actual Expense

Products / Materials




R 5 000.00


Malt Extract Agar (MEA)

R 313.00


Standard 1 Agar (STD1)

R 300.00


Petri dishes

R 7 000.00


Blue Pipette Tips

R 359.00


Yellow Pipette Tips

R 359.00


White tips (molecular)

R 359.00


Ringer's Solution

R 226.00


Nucleospin Plant II

R 8 647.00


SeaKem LE Agarose

R 400.00


Flat Cap PCR eppies (Genuine Axygen Quality)



Ethidium Bromide



FSpBI (BfaI) enzyme

R 952.00


Tango Buffer



McCartney bottle and rubber cap



Primer 1 (Its)



Primer 2 (β- tubulin)



Taq DNA polymerase (Bioline)

R 4 661.39


Magnesium Chloride

R 138.00


PCR buffer blue (5x)



Tris (Base)

R 485.00


Boric (Acid)

R 139.00


EDTA (Free acid)

R 102.00


Loading dye



Hyper Ladder 1(Bioline)

R 1 753.78


Power-free Nitriles examination gloves (Microflex)*



Administrative Expenses









Office Supplies

R 100.00





Travelling Expenses



Mileage (R5.52pkm) (Local Market)

R 1 589.76





Flight & Accommodation (3 trips - Ceres)

R 8 000.00


Total Expenses

R 40 883.93


*Awaiting feedback from suppliers


The beneficiaries from this research experiment will be the DFPT (Deciduous Fruit Producers trust) industry, farmers, pack house and local fresh produce market.