17.19 Production of spores and other inocula

Fungi are able to parasitise organisms from the other eukaryotic kingdoms and have become recognised as biocontrol agents of pests, parasites and pathogens of various sorts. Pathogens of weed plants may be harnessed as mycoherbicides (see Chapter 14; CLICK HERE to view the page), and mycopesticides can be formulated from pathogens of animals (see Chapter 16; CLICK HERE to view the page), including nematodes (discussed in Chapter 15; CLICK HERE to view the page).

We have also mentioned before the many species of fungi which are parasites of other fungi (mycoparasites) and which might serve as biocontrol agents of pathogenic fungi. Trichoderma spp., which are common in soils are particularly interesting because they are opportunistic parasites of other fungi; especially of common diseases like Rhizoctonia damping-off of seedlings and Armillaria root diseases of trees, as well as being potential symbionts of plants (Harman et al., 2004)(see the section Mycoparasitic and fungicolous fungi in Chapter 16; CLICK HERE to view the page). And then there are all those mycorrhizal mutualistic associations between fungi and the roots of plants (see Introduction to mycorrhizas in Chapter 13; CLICK HERE to view the page) that could be developed and exploited to improve growth of crop plants without chemical fertilisers. The mycoparasitic and antagonistic fungi are a potential biological alternative to chemical pesticides for the control of weeds and pathogens (Hussain et al., 2012).

In all of these cases the potential commercial product is the fungal biomass, in the form of mycelium and/or spores, and fermentation offers a practical method of producing this. For example liquid fermentation has been used to produce conidia of Trichoderma harzianum in bulk to control fruit rot diseases caused by Botrytis cinerea (Latorre et al., 1997). Conidia were produced in a 300 litre fermenter operated at 28°C for 60-70 h with an aeration rate of 0.4 volumes of air per volume of medium per minute and a rotor speed of 180 rpm, with constant low level illumination (Mendoza-Mendoza et al., 2016).

Similarly, conditions for optimising chlamydospore production, by mycoherbicidal strains of Fusarium oxysporum (Ascomycota) have been studied with 2.5-litre fermenters, and subsequently scaled-up to 20-litre (Hebbar et al., 1997), demonstrating that a single step liquid fermentation could produce large numbers of chlamydospores (about 107 ml-1) in less than 2 weeks. Chlamydospores are more resistant to desiccation and temperature extremes than other spore types and are therefore easier to formulate into mycoherbicides that have the necessary shelf life for a commercial product. Chlamydospores of Trichoderma spp., which are also widely used biocontrol agents antagonistic to a variety of plant pathogens, are similarly highly resistant to adverse environmental conditions and therefore have improved shelf life but their large-scale production has proved to be difficult. Peng et al. (2021) carried out a comprehensive analysis of the transcriptome in Trichoderma to establish the molecular mechanisms central to chlamydospore formation, covering eight different developmental time points in the growth of mycelia. They found enhanced expression of genes relating to glycogen, lipid, mannan, and chitin synthetic metabolic pathways during chlamydospore formation. In one strain of T. virens, the chitin synthase gene was the key gene of the pathway; when this was deleted the mycelium was unable to form normal chlamydospores.

As a final example we will mention the induction of submerged conidiation of Ulocladium atrum (Ascomycota) which has potential for controlling grey mould caused by Botrytis cinerea in glasshouse and field-grown crops. Yields of maximum total inoculum (mycelial fragments and conidia) were approximately 2×107 ml-1 after 9 days incubation at 25°C with a 100 rpm rotor. Shelf-life studies showed that viability was comparable to that of aerial spores from 4-week fermentation on oat grains. However, the ability of submerged biomass to germinate in drier conditions declined significantly after 6 months; no such decline was observed in aerial spores formed on solid substrates (Frey & Magan, 2001).

This last example emphasises the fact that the properties of spores produced in solid state fermentations differ distinctly from those obtained in submerged liquid fermentations. Fungal spores used as biocontrol agents are produced preferentially in solid state fermentations because the spores obtained are of higher quality; meaning specifically that they are more resistant to desiccation and are more stable in a dry commercial preparation, though they also display morphological, functional and biochemical differences compared to those produced in submerged cultures (Feng, Liu & Tzeng, 2000; Hölker, Höfer & Lenz, 2004). The best yields of spores are produced by a combination of submerged liquid fermentations (for biomass production in a first ‘seed’ step) and solid state fermentation (for subsequent spore production).

Substrate types are extremely varied and include all sorts of seeds and cereal grains as well as many waste plant materials (see the section on Solid state fermentations below; CLICK HERE to view the page immediately). In some fungi (for example Coniothyrium minitans a biocontrol agent active against the plant pathogenic fungus Sclerotinia sclerotiorum) spore production only occurs at the surface of the substrate (Reddy, 2013).

Spores for applications in the food industry have often been produced as starter culture by solid state fermentation because it gives better yields of homogeneous and pure spores. This applies to Penicillium roquefortii, P. camembertii, used in the production of blue cheese and brie-type cheeses, and P. nalgiovense which is used in the production of salami (see the section on Cheese and salami manufacture; CLICK HERE to view the page immediately). Although they can be grown in submerged batch cultures, industrial spore production of these Penicillium spp. tends to favour solid substrates (bread, seeds, etc.) that result in yields of up to 2×109 spores g-1 substrate in a 14 day cultivation, which is approximately ten times the yield that can be achieved in submerged liquid cultures. We will deal with solid state fermentation in more detail below (CLICK HERE to view the page immediately) after a brief look at the planet’s biggest and most widely distributed submerged liquid fermentation process.


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Updated June, 2021