The Sweet Taste of Malt and Revealing its Pathogen’s secret

The Sweet Taste of Malt and Revealing its Pathogen’s secret

A Flame-Side chat about how international travel, collaboration and interaction led to new insights in pathogen biology inside (malted) grains

Dear brew enthusiasts,

Last week, we discussed a paper from researchers at North Dakota State University who investigated the persistence of Fusarium on grains and how that can impact mycotoxin buildup during the malting process. They recently had their work accepted which you can now also find here (for a full reference, see below).

I am delighted to present you with a flame-side chat with Professor Paul Schwarz, Dr Shyam Solanki and Dr Zhao Jin. In this interview, we will delve into the reasons for their investigation, the extent to which mycotoxins cause problems in malt production and of course, what questions they now wish to address!

Prof Paul Schwarz taking notes at the NDSU’s barley FHB screening nursery in Nanjing China. (Source: Prof Paul Schwarz)

A little background about the research team

While Prof Schwarz is a cereal chemist, he has enjoyed working closely with barley breeders and pathologists.  North Dakota began having issues with FHB in the early 1990s, and he shifted much of his work to mycotoxins.  As a result, he has made more than 30 trips to China for either FHB-mycotoxin or brewing related work.

Paul first met the future lead author, Dr Zhao Jin,  on one of these trips when she was an MS student in Fermentation Engineering at Jiangnan University. She eventually became a visiting scientist and then a post-doc in his lab.

Dr Zhao Jin working at the NDSU’s barley DON lab in Fargo ND USA (Source: Zhao Jin).

Zhao’s research mainly focuses on the “Characterization of trichothecene mycotoxin development during the malting of Fusarium infected barley and other grains” during she works with Dr Schwarz at NDSU. A big challenge in this research is to observe the fungal localization, particularly the ingrowth of grain tissues. Dr Shyam Solanki’s post-staining method provided key technical support to achieve the objective of hyphal growth tracking in FHB infected grain and malt kernels. Shyam was a pathologist/research scientist at Prof Robert Brueggeman’s Lab at North Dakota State University when they had this collaboration.

Dr Shyam Solanki, Postdoctoral researcher at North Dakota State University. Source: Dr Shyam Solanki.

This wonderful backstory speaks volumes about the value of both cross-discipline, local and international collaboration in science (and beyond).

Let’s dive in!

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Edgar: In your MS, you allude to the observation by some maltster that despite low level of Mycotoxin contamination in raw materials, higher levels are occasionally found in malted grains. Could you elaborate on the extent of this problem currently?

Paul: This type of  Fusarium growth and mycotoxin production in malted barley is a problem that is seen only in some crop years, and then only in certain growing locations. Speaking with friends in the industry, the last significant occurrence of this issue in North America was in 2018, in some growing regions.

Nevertheless, if we think of the scale of some of the larger commercial maltings, these “problem samples” can represent a significant monetary loss in terms of grain and production time. In general, maltsters have gained considerable experience in handling Fusarium infected grain. When samples with low DON, and presumably surface infection are selected, and then stored for 6 or more months, malts with little to no DON generally result. One maltster has told me that knowing the location of infection would be useful, as the “internally” infected samples still present problems, even after storage.

Zhao: In addition, a recent worldwide survey of more than 1,000 commercial beers (produced during 2011-2014) collected from 47 countries showed that Deoxynivalenol (DON) and its plant metabolite DON-3-β-D-glucopyranoside (D3G) were the major mycotoxins detected and attributed to Fusarium infection. The level of mycotoxin (DON+D3G) was found to range from 10 to 475 μg/ L in 406 beers, of which 73% were craft beers [1].

The grain bill of craft brewing usually contains not only malted barley, but also wheat, rye, triticale, and/or their malt as specialty malt to enhance beer flavour. However, the concern is that these adjunct grains and malts might be sources of mycotoxins, such as DON. 

Edgar: You show that during malting, both the levels of Fusarium and DON increase on all grain types (though at different extents). Is there a strict correlation between biomass and DON levels or do you think that there is a temporal/and or spatial aspect to DON metabolism? Is the pathway stage-specific or activated by particular signals?

Zhao: Generally, practical experience has shown that FHB infected barley with low DON levels (e.g. <0.5 mg/kg) can often be used in malting as DON declines during steeping and remains lower on malt. However, an occasional problem is that germination of lower DON barley sometimes results in malt with DON levels that are higher than the original barley.

In our last research project, increases as high as l0-fold were observed during the malting of wheat, rye, and triticale that had relatively low initial DON levels. The goal of the current research was to find out the reason (Fusarium hyphal localization on/within kernels) for the malt DON increase, and thus, we only selected the several grains and corresponding malt samples representing the aberrant behaviour where the levels of Fusarium and DON increased dramatically during malting.

The Fusarium biomass was found to have moderately high correlations with malt DON levels when we investigated malted wheat and rye samples.  The biomass of Fusarium was quantified with toxigenic Tri5 DNA concentration by using quantitative-PCR, but we should keep in mind that qPCR can’t be able to detect the viability of Fusarium

In terms of temporal/and or spatial aspect, we found hyphae mainly localized in the husk and vascular bundles of barley. However, the growth has a chance to expand into the interior of kernels, such as the aleurone layer, endosperm, and embryo tissues following malting. This type of “internal” infection was found to commonly exist in wheat, rye and triticale grains.

Particularly, cavities that usually located in the center of the transverse section of these grain kernels were filled with hyphae. Hyphae were observed to distribute much heavier in malt kernels of wheat, rye and triticale. Obviously, cleaning and steeping operations are not easy to get access to the removal of hyphae in these “internal” regions and they are potential sites of Fusarium growth and mycotoxin production during the germination stage and withering of kilning. 

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Shyam: That is an interesting question, initial stages of malting such as grain germination provides a conducive environment for Fusarium growth, so I can speculate that the increased mycotoxin should correlate with the extended growth of the pathogen at this time. However, we need to investigate further to see if the DON metabolism varies depending on the malting stage or presence of the pathogen in certain layer/s of different kernels.

Edgar: You used both staining protocols as well as molecular approaches to detect F. graminereum. What, in your opinion, is the best approach to detect this pathogen and guard against F.g. contamination and growth in a commercial setting?

Shyam: Each technique has its own merits and pitfalls. In commercial settings, I would assume that the primary goal is to determine the presence and levels of Fusarium rather than its localization and distribution. Thus, a semi-quantitative or quantitative PCR based approach or isothermal amplification such as endpoint loop-mediated isothermal application (LAMP) would provide a rapid, cheaper and more accurate detection of pathogen species.

In fact, we used next-generation sequencing technology available in-house in Dr Brueggeman’s lab to identify Fusarium graminearum abundance in the samples with the expertise of Dr Sharma Poudel and Dr Ameen to conduct the bioinformatics analysis.

However, these traditional amplification and sequencing techniques would not be able to give us information about the site of infection. For example, if somebody were to be interested (as Prof Schwarz mentioned before) in the spatial distribution of fungal species in the epidermis, testa, pericarp or other internal compartments of malting grains, then it is necessary to track the pathogen growth during the malting process using the fluorescent dyes.

A staining protocol can precisely inform us about the location of the pathogen, which was important in our study to determine the cause of the increased amount of mycotoxin in malting grain due to surviving Fusarium.

However, post staining protocols could not differentiate between the fungal pathogen species or genera and you can’t precisely determine if visual hyphal growth belongs to Fusarium or other filamentous fungi. Furthermore, a gas chromatography method can provide accurate information about the mycotoxin itself, but it is time-consuming and requires technical expertise. Thus, we harnessed the power of all these approaches to identify how the Fusarium hyphae expansion during the malting process leads to increased mycotoxin production.

Edgar: We know that Fg growth on or in grains, is responsible for high mycotoxin levels in malted grains. Do we know how infection of grains affects the overall quality with respect to malting?

Paul: Beer gushing is probably the best-known quality defect in using “moldy” grain. This is the eruptive, or spontaneous over-foaming that can occur upon opening a bottle of beer. Infection with a number of genera of fungi, including Fusarium, has been associated with the production of hydrophobins, which are one cause of gushing.

Colonization of grain is also associated with the production of fungal enzymes. F. graminearum is known to produce cell-wall degrading and proteolytic enzymes. Often seen impacts of infection on malt quality are, increases in malt soluble protein, free amino nitrogen and wort colour.

Edgar: The availability of new methodologies that now allow detection of fungal diseases in situ, opens up new avenues for future research into the management of the disease, what do you think pathologists should focus on in our bid to better control this pathogen in the field?

Shyam: Genetic control is the most sustainable source of controlling plant diseases. Staking of multiple resistance genes and testing their efficacy in multiple agroclimatic zones is important to achieve a durable and broad-spectrum pathogen resistance is a better control strategy coupled with other cultural practices. In fact, the development of synthetic immune receptors and their incorporation in crop enhancement programs to achieve diverse-pathogen resistance could be a game-changer in the future. However, genetic control is not always available and might bring linked undesirable quality traits. The second best and readily available control measures include use of fungicides and pesticides, which provides an excellent solution against the damaging pathogens in the field setting, and farmers should use them judicially with other integrated disease control strategies.

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Edgar: What were the major problems or bottlenecks that hampered your work?

Zhao: One of the bottlenecks is that we only use slides of grain/malt tissues with 5-10 μm of thickness because starch is not penetrated by either electron or laser. The whole kernel examination is not realistic according to the current technology development. Another one is that qPCR and microscope can’t detect the viability of microorganisms. False-positive results would be generated if used to screen samples.

Shyam: As mentioned by Dr Jin, thin sections of grain/malt were used for staining purpose, so most of the times there was a problem of tissue detachment and endosperm layer washing off during the multiple staining steps.

Edgar: What do you think the interesting questions are from this work that you are now pursuing?

Zhao: From this piece of research, we found that the fungal localization within barley is very different from wheat, rye and triticale grains. This discovery is very important because it affects the fate and production of mycotoxins during malting. The behind question is what caused the difference? The grain structure and Fusarium infection timing are two potentially key factors.

In terms of structure, the barley husk serves as a barrier to infection. However, the husk threshes free in wheat, rye, and triticale. The infection timing is of interest because wheat, rye and triticale flower after heading, while barley flowers in the boot stage.

Rye is also open-pollinated and flowers for an extended period of time. These differences in the flowering pattern could lead to more extensive colonization in wheat, rye and triticale. In the following research project, we have simulated the early and late infection of barley in the field to find out the effects of infection timing on the Fusarium growth and mycotoxin production during malting.

Shyam: From a researcher’s perspective I would like to identify the genetic factors that control pathogen growth in certain grain types so we can use them to device better pathogen management strategies.

In short term, I would also like to test if the presence of Fusarium mycotoxin is uniform or differentially compartmentalized in the infected malting grains?

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Thank you so much for providing such great insight into your work. I hope (and I am sure) that this Flame-Side Chat and our post will have convinced our readers on how important it is to use malts that meet the highest standards and be aware that raw or malted speciality grains need to be of high quality (and stored appropriately). A heartfelt thanks to Paul, Zhao and Shyam for their thoughtful and insightful answers. Any More questions about this or related work? Get in touch.

References:

[1] Peters, J., Van Dam, R., Van Doorn, R., Katerere, D., Berthiller, F., Haasnoot, W., & Nielen, M. W. (2017). Mycotoxin profiling of 1000 beer samples with a special focus on craft beer. PloS one, 12(10), e0185887.

[2] Expansion of internal hyphal growth in Fusarium Head Blight infected grains contribute to the elevated mycotoxin production during the malting process. Dr. Zhao Jin, Dr. Shyam Solanki, Dr. Gazala Ameen, Mr. Thomas Gross, Mr. Roshan Sharma Poudel, Dr. Pawel Borowicz, Dr. Robert S. Brueggeman, and Dr. Paul Schwarz. Molecular Plant-Microbe Interactions®: https://doi.org/10.1094/MPMI-01-21-0024-R

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