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A veterinary detective story

This veterinary detective story was written by Dr. Carolyn Orr of the Animal Clinic of East Avenue in Brockport, New York. It is a companion to our story, Eulogy for a Lost Dog, which tells the tale of Shiloh, a young dog who died of Galerina mushroom poisoning. Here Dr. Orr tells the same tale from a rather more technical, veterinary perspective, revealing the sleuthing behind this perplexing case.

Acute liver necrosis and death from Mushroom toxicosis in a young Great Pyrenees: A Veterinary Perspective

Dr. Carolyn Orr, DVM

Shiloh was a 13 month old female, spayed Great Pyrenees; one of our patients. On Monday morning we were informed that she had presented to a local emergency clinic the previous day with vomiting and icterus (jaundice).

Shiloh was a healthy young dog showing a normal growth rate. She had undergone routine anesthesia for an ovarian hysterectomy at six months of age with no unusual complications, and had received all scheduled vaccinations (including that for Leptospirosis: four-way from Fort Dodge) and routine antiparasitic medications as a puppy, including heartworm prevention. One complicating factor to diagnosis was that she had gotten a bit finicky about her diet during the two months prior to this incident, but she showed no other clinical symptoms and no weight loss.

On Monday morning, the emergency clinician reported Shiloh as stable, but indicated that she had experienced two episodes of vomiting on Sunday, plus severe jaundice. Shiloh ate while hospitalized Sunday night and had stopped vomiting. Monday morning, the emergency clinician believed she was fit to return home, but recommended she be transferred first to me for examination. When Shiloh presented at our clinic at about noon, her condition had worsened notably from her discharge condition just hours earlier. She had been described as energetic and playful on Saturday, had vomited only once Saturday night, with progressive inappetance and more vomiting Sunday. On presentation on Monday, she was barely able to support her own weight for more than 2-3 minutes, yet still wagged her tail in acknowledgment and greeting.

Records from the emergency clinic showed a normal examination on Sunday, except for the icterus (temperature of 101.7 degrees F, heart rate 96 bpm, respiratory rate 36 bpm). Shiloh was comfortable on abdominal palpation and showed pink but icteric mucous membranes. Blood work revealed slightly elevated HCT 50%, WBC 16,600 with normal distribution; blood chemistry is shown in Table 1. A leptospirosis titer (7 serovars) had been sent off for evaluation to Cornell University’s Veterinary Clinic/Lab but results would take 3-5 days. She had been started on ampicillin (an antibiotic effective against leptospirosis) and famotidine (an H2 blocker antihistamine useful in cases of stomach irritation) and Normsol IV fluids. The report indicated that she had not eaten anything further after Sunday night.

At approximately noon on Monday (approximately twelve hours later), Shiloh presented to our clinic. She was weak and lethargic with moderate and variable pulses, notably icteric sclera, roof of mouth and skin. This was very different from her description upon discharge from the emergency clinic. She was notably not pale on her mucous membranes. Her temperature was 100.4 degrees F, heart rate 116 bpm, respiratory rate 28 bpm. We hospitalized her, resumed injectable antibiotic and repeated blood work (see Table 1). Her initial blood work (Sunday night) had many values that were within normal levels, but that showed potential abnormalities upon closer analysis. Her repeat blood work showed deterioration consistent with her clinical presentation, and with more dramatic changes than common diagnoses such as hepatitis, cholangiohepatitis, cholestasis, or liver toxins more routinely observed in dogs. Blood chemistry is shown in Table 1; however, Alb, GGT, and Crea could not be determined due to the high level of icterus (see Wikipedia’s discussion of liver function tests for more insight, or Vet Tech’s brief explanation of Blood Test Results).

	HCT	WBC	ALT	AKP	TP	Gluc	Alb	GGT	BUN	Crea	T.BIL	platelets
Sunday	50%	16600	1345	1096	98	68	3.0	15	5	1.1	9.8	–
Monday	39%	20680	849	718	–	69	–	–	3	–	14.8	264

IV fluids were continued with added dextrose, antibiotics, H2 blockers, and Vitamin K injectable. I further ordered an ultrasound to rule out gall bladder obstruction—problems with the gall bladder can often be associated with vomiting and jaundice. However, the ultrasound was normal with only note of a mild coarseness to the liver in general but otherwise normal echogenicity and borders. No other abnormalities were observed on ultrasound.

At this time I felt we had ruled out numerous problems but still had no control over the disease process. I discussed many potential toxins with the clients, but they had not observed any definite ingestions. The highest likelihood diagnosis on our list other than a toxin was leptospirosis, a bacterial disease that might be consistent with Shiloh’s symptoms. We have experienced occasional cases caused by the uncommon (in dogs) serovars Leptospira autumnalis and Leptospira bratislava in the recent past in the area. I indicated my concern to the client with Shiloh’s rapid and progressive deterioration, especially the deterioration of functions like glucose production and low BUN levels. If we supported the diagnosis of leptospirosis I would not expect clinical improvement for another 24+ hours from antibiotic therapy and gave a very guarded prognosis. Unfortunately, she continued to deteriorate quickly and fell into semi-consciousness with head tremors, lateral recumbancy, poor responsiveness with eyes rotated caudally except with stimulus. She continued to be very icteric with developing signs of petechiation of ventral abdomen, mild ascites, hypothermia, poor thready pulses, and tachycardia.

At this time, our clients made the difficult decision to euthanize Shiloh due to her rapid deterioration, progression to neurologic signs and poor prognosis for recovery. I requested a gross necropsy and pathology of liver and kidneys for the potential better understanding of Shiloh’s acute illness and death.

Shiloh had gone from a normal liver (blood work at 6 months was normal) to acute liver failure in 36-48 hours. Leptospirosis was still highest on the list of differentials. It occurred to me that the titer drawn on Sunday would most probably be negative because Shiloh would not have had time to seroconvert (develop antibodies) if she had recently acquired the infection. Therefore I sent a sample of the liver to Cornell for a leptospirosis fluorescent antibody test. I also saved a small sample of the liver for possible toxicology testing, and sent a sample of liver and kidney to a pathologist for evaluation. Leptospirosis titers later came back negative, and the fluorescent antibody test was also negative. Leptospirosis was not the cause of Shiloh’s rapid decline.

Liver pathology confirmed that liver failure was due to severe hepatic necrosis of a type that could be caused by amatoxin-containing mushrooms. Some mild renal (kidney) changes were also observed that might have arisen due to recent acute dehydration, but which were also consistent with mushroom poisoning. One determining observation was the lack of the fatty liver changes that had been seen with aflatoxin cases due to dogfood contamination in 2005. We could rule out aflatoxins as a cause.

The highest causation of the pathological findings was mushroom toxicosis: the liver histology was consistent with poisoning by mushrooms containing amatoxins. Even though we had no observation of mushroom ingestion, after talking with Ms. Munganast she did remember mushrooms growing in the yard. She had never observed Shiloh eating the mushrooms, but Shiloh had a history of chewing on things. An early attempt to identify the mushrooms from photos suggested they were not toxic. However, later examination at Cornell confirmed the mushrooms were a species of Galerina that tested positive for amatoxins with the Meixner test. Thus we were finally able to determine that the probable cause of Shiloh’s death was mushroom poisoning.

You can find Shiloh’s story, as told by her owner, right here.

Eulogy for a lost dog

This post was written by our guest Tami Mungenast in remembrance of her beautiful dog, Shiloh. Thanks for sharing your story, Tami.

Have you ever heard of a pet soul mate? I know some of us believe in the idea of having one true soul mate in a romantic relationship.  I also experienced what it was like to have a soul mate in a pet relationship. Her name is Shiloh. She was a Great Pyrenees who we adopted into our family as a young puppy. I actually felt the connection to her before she was conceived. After meeting Shiloh’s parents I felt the strong need to have a pup from this set of parents. I anxiously awaited for the heat cycle and kept close tabs on the pregnancy of Shiloh’s Mom. When the litter was born on July 8th 2007, I was given first choice of the pup I wanted. I instinctively knew that I should have the first born to these parents. The decision was made even before the pups were walking. It was just a strong feeling that I had knowing that the first born was meant to be part of our family. That was Shiloh. Shiloh quickly became a loving and devoted family member. She was a dream pup in the sense of her easy going behavior and laid back attitude. She grew quickly and was expected to reach 115 pounds by maturity. Though intimidating in size was she was really a gentle giant. While protective of her family and reserved with strangers she was quite the teddy bear at heart.  

Shiloh was 13 months old when we lost her. I never saw it coming. The thought of losing her so soon never ever crossed my mind. Her health was excellent. I worked closely with my Veterinarian Dr. Orr on any health care issues. She was given daily walks, exercised regularly, brushed almost daily, weekly playdates with other neighborhood dogs, lots of love and interaction with our family including our two young daughters. In return she gave us 100% loyalty and devotion. She was eager to please and was extremely well behaved. She even took first place at her dog obedience classes–a reflection of her temperament and not our training ability.  

On Friday August 15th we came home late after bringing the kids to the Jonas Brothers Concert at Darien Lake. When we got home we noticed that Shiloh had vomited in her pen. The vomit was a very small amount of yellowish spit and did not set off any alarms in my head at that point. It was so late that we let her in and brought her up to bed with us. The next morning Shiloh was very disinterested in her food. I realized that her food bowl was still full from Friday, and her water bowls were not empty as they usually would be in the morning. Thinking back to that week I did recall that she vomited midweek a very small amount of half digested carrots that we gave her. Later that morning and during the early afternoon I did my usual running around with the kids. One daughter had horseback riding lessons and the other had a friend sleep over the night before who I needed to get home.

After I came home I realized that Shiloh still had not eaten her food. Now I was concerned that perhaps she was beginning to show the signs of an intestinal blockage. Shiloh had a powerful jaw and a strong desire to chew hard things. She loved to chew sticks and branches and any hard plastic toy she could get. We were pretty good at keeping dangerous items from her reach but with young children at my house there was always the chance that she gotten someone’s toy. So I made a phone call to our vet to have her seen as soon as possible after the weekend. The situation did not seem to be an emergency, but I was trying to be proactive for the possibility that she might be developing some type of obstruction. Later that day I enticed Shiloh with a can of dog food–she wolfed down the canned food with no problem. When she went outside one of the kids noticed that her urine was darker than usual. This caused me some concern but I suspected that not eating or drinking well for the past 24 hours could be causing her urine to be more concentrated. I checked her gums for hydration and they were not as colorful as usual but looked ok. We spent the rest of the day at home. Shiloh was doing her usual guarding of the property and sounding off at the neighbors who were shooting off fireworks. Later after dinner we took a family walk with Shiloh. Little did I know this would be our last family walk with her. She strained to have a bowel movement when we got back home. This made me feel even more concerned about a possible obstruction. We brought her to bed with us that night, feeling uneasy.  

On Sunday morning I woke up at 6 am to get ready for work, and Shiloh and I went downstairs together. As soon as she got down the stairs she began vomiting large amounts of the canned food that she had eaten the day before. I instantly felt panicked and called the emergency veterinary hospital, telling them I was concerned about a possible blockage. They told us to bring her right in. After I hung up I took a closer look at Shiloh’s face and realized that the whites of her eyes were now yellow. I began to panic. At the emergency clinic she was given supportive care. Tests they ran showed liver problems–there was no intestinal blockage. They ran various blood panels and began treating her for a possible Leptospirosis infection. Even though she had been vaccinated against Lepto, the veterinarian felt this was a strong possibility, but test results would not be back for several days. They hospitalized Shiloh. I called every 3-4 hours to check on her status. The staff seemed calm and hopeful that she would be fine.

On Monday morning I asked that she be transferred to Dr. Orr, my regular Vet, for further treatment and a conclusive diagnosis. My husband picked up Shiloh as I waited at Dr. Orr’s office. When she walked into Dr. Orr’s office I fell apart. I could not believe the condition she was in! I sat on the floor hugging her and crying while she rested her head on my shoulder. She looked so much sicker than she had the day before. Her condition had deteriorated horribly and I was shocked. Her gums, eyes and ears were very yellow now and she was extremely lethargic. I was given some time with her before I turned her over to Dr. Orr for more tests. I went home and for a few hours anxiously awaited news on Shiloh. Dr. Orr called later that afternoon to let me know that Shiloh was in very serious condition. Her liver was failing and she was not responding to the antibiotics prescribed for Lepto. Dr. Orr said that she needed to respond soon otherwise the outcome would not be good. She invited us back to her office to spend time with Shiloh. My husband, daughters and myself went back to Animal Clinic of East Ave to see her. They gave us an examining room where we could spend alone time with Shiloh, who lay on the floor near us with her IVs. It was unbelievable to see such a healthy vibrant dog turn so grave in such a short time. It felt like a bad dream that I could not awake from. The staff brought Shiloh a blanket to lie on and gave us as much time with her as we wanted. They even brought the ultrasound machine to us so we could stay together during this procedure. Dr. Orr warned us that Shiloh was running the risk of DIC. She explained that Shiloh’s platelet counts were dropping and she could bleed out during the night. If this occurred there would be nothing she could do to stop it.

It was now 10 pm and we did not want to leave Shiloh but the office had closed at 8 pm and I needed to let the staff go home. The kids were becoming restless as well. I took Shiloh out for her last walk. She barely had the strength to walk but was able to manage to get outdoors. As soon as we got to the parking lot she drummed up enough energy to start pulling me towards my Jeep. She wanted me to take her home. I felt good about seeing her spirit still there and her eagerness to come home. I left hoping and praying that she would make it through the night and her body would start to respond to the antibiotics. I gave her a big hug and kiss before I left and asked her to hang in there and not to give up. We all told her we loved her and went home. The night passed into the morning with no phone calls. I was feeling hopeful that her condition could be improving.  

On Tuesday morning I called at 7 am to check on her. The technician said that her condition had worsened and Dr. Orr was on her way in. Shortly after Dr. Orr called me to tell me that Shiloh had slipped into a coma and had about 4 hours left. Her liver had failed and there was nothing they could do. I could not even talk after receiving this call. I needed to hang up the phone to try to get a handle on this devastating news. This was not supposed to happen–she was too young and too healthy to die. She was such a good dog and did not deserve this outcome. For the past year I’d tried to do everything right with her–how could this be happening? How could she be leaving us already? The thought of her lying alone in a cage suffering and waiting to die was unbearable. So I asked my husband to go be with her and have her euthanized to prevent those final hours of suffering. He was upset as well, but I was completely shattered and could not even speak clearly. I was feeling the worst pain I had ever experienced in my 40 years of life. I knew that I could not keep it together and handle being there for the euthanasia. I was emotionally and physically sick. My husband was generous enough to take on this responsibility for me.

After Shiloh’s death, Dr. Orr suggested we run some tests on Shiloh’s liver to help us better understand what caused her sudden death. The feeling was that it was Lepto, but we still did not have the test results back. Plus we still had a dog at home and needed to keep him safe: We had taken in Shiloh’s baby brother to foster in early July after their Mom was killed by a truck. He was only 10 days old when we took him in to care for him. With all of the possibilities of what could have caused Shiloh’s death we discarded all of Shiloh’s food and treats. We also kept the puppy away from other dogs in case it was an infectious disease like Lepto, which can be spread through contact with urine. We waited anxiously for the necropsy results. I spent endless hours researching her symptoms and going over every step I took with her during her final week. I rethought every decision I had made about food choices, walks, puddles, water contamination… everything. The overwhelming concern that something lurking on my property had caused the sudden death of our 92 lb healthy dog was very draining. I was suspicious of everything. 

The following week the Lepto test came back negative. Dr. Orr asked us to be patient and wait for the pathology report on the liver for more answers. The pathology came back two weeks after Shiloh’s death, and indicated that it was a toxin that caused Shiloh’s liver to fail. The pathologist felt that toxic mushrooms should be a primary suspect. We have had mushrooms pop up in our yard from time to time, and this rainy summer they had been abundant. We never worried much about the mushrooms and generally just mowed over them. I never thought the deadly mushrooms you hear about were something that would grow in a suburban yard. Ironically, the day Shiloh became ill my husband had picked a few mushrooms from our yard where Shiloh often laid. He put them in the garage and forgot about them until we had got these results. Dr. Orr recommended that we have the mushrooms identified. I honestly did not think that mushrooms were the cause of Shiloh’s death but took Dr. Orr’s advice and contacted a local mushroom expert. He asked me to take some pictures of the dried specimens and email them to him for possible identification. Upon receiving the (out of focus) pics and talking to me about our yard he felt that the mushrooms were likely not the cause of Shiloh’s death, but he wasn’t able to identify the mushrooms in the photo. I contacted Dr. Orr with this news and she urged me to send the specimens to a fungal expert at Cornell University–Dr. Hodge. I contacted Dr. Hodge and she was generous enough to spend time talking with me and agreed to look at the dried mushrooms we had. They were shipped to her for further analysis.  

I’ve dreamed of Shiloh often since her death. In my dreams she was sick, her eyes were yellow and she was dying. I even dreamt that the pup, Shiloh’s brother, was dying. On one occasion I dreamt that my own liver was failing. I would go to sleep thinking of Shiloh and mourning her, and I would continue to dwell and dream on her all night long. On Wednesday Sept 3rd I awoke to feel Shiloh nudging me with her nose. She often did this during the night when she felt the need for some attention. I woke up and felt her presence in my room and felt eerie about the nudge that woke me from my sleep. I looked at my husband and he was still sleeping. I finally fell back asleep to find Shiloh in my dreams again. This time she was not sick. She was healthy and vibrant. I was petting her and hugging her, feeling so happy that she was still with me. My dream felt so real that she was with me….I could touch her and feel her but then I realized that I could not smell her. In my dream I kept sniffing her wondering why I could not smell her and wishing that I could. In the morning I woke with the strong feeling that Shiloh had visited me during the night. But I could not make sense of the dream. Later that morning Dr. Hodge called to tell me that the mushrooms I sent from my yard were Galerina mushrooms and tested positive for amatoxins. Given the toxicity of the mushrooms it would not have taken many to cause Shiloh’s death. 

Dear Tami,

The three brown mushrooms you sent me are a species of Galerina. You mentioned that you collected them around the time Shiloh became ill. I am unable to quickly identify them to species, because there are very many Galerina species and they are difficult to distinguish–even if I had had fresh specimens in hand. But I believe that identifying the species of Galerina is not important in this case in light of the test results I describe below.

Many Galerina species contain a powerful family of toxins called amatoxins (or amanitins). They are the same toxins found in Amanita mushrooms (the death cap and the destroying angel). Amatoxins typically cause vomiting and diarrhea in early stages, some hours after consumption. These symptoms often appear to remit, but the toxins are meanwhile destroying actively metabolizing tissues, particularly in the liver. There is no specific antidote, although administering activated charcoal in early stages can help remove some toxins from the intestinal tract. Human survival rates hover around 60%, from what I’ve heard, and survival depends on both the amount eaten and on early diagnosis leading to appropriate supportive care.

I performed a Meixner test on the brown mushrooms. It’s a crude test for amatoxins that depends on their interaction with the lignin in newspaper when exposed to hydrochloric acid. It is not definitive–only suggestive. A blue reaction in the Meixner test suggests that amatoxins may be present. I’ve attached a photo showing the strong blue reaction I obtained from the brown mushrooms. They are potentially fatally poisonous.

Sadly, there’s no reliable way to remove these mushrooms from your lawn. You can remove the mushrooms themselves (requires close attention to find them all), but the underground mycelium will persist from year to year. Some have suggested treating the lawn with lime to make it inhospitable to the mycelium, but there’s no evidence that this works. Note that that you can’t be poisoned by touching the mushrooms–only by ingesting them, raw or cooked.

Lastly, and I bet you’ve done this already–I suggest you have a very serious talk with your kids about mushrooms.

Kathie T. Hodge

I spoke with Dr. Orr about the news of the mushrooms in my yard. Since I never actually saw Shiloh eat a mushroom I was having a hard time believing this could be the cause. After a lengthy talk with Dr. Orr, and based on what the experts saw in Shiloh’s necropsy, plus the fact that these deadly mushrooms grew right where Shiloh often lay, Dr. Orr felt it was conclusive that they were what caused Shiloh’s death. 

Looking back now I feel that Shiloh’s death served a greater purpose. I had something very deadly lurking in my yard. This posed an unseen risk of death to both people and animals. My yard is the one where all of the neighborhood kids play–children from 1 to 11 years old. The possibility that some child could have ingested a bite of these deadly mushrooms was certainly there. In addition to the kids, we were caring for Shiloh’s baby brother Sampson who is a typical curious pup and puts everything in his mouth. He certainly could have come in contact with these Galerina mushrooms and might have died as well. The loss of Shiloh, my pet soul mate, brought our attention to the deadly fungus growing in our yard. Shiloh lost her life but her death may have saved others’ lives.

In addition to being neurotically cautious with any mushrooms in my yard now I have also spread the word of Shiloh’s story by contacting news organizations, which have run stories and TV broadcasts on Shiloh and the dangers of poisonous mushrooms. I have also spread the word through various dog and Pyrenees groups that I belong too. I have received many phone calls and emails from pet owners and parents thanking me for sharing my story. They are now more cautious with mushrooms in their yard and are prudent on educating their children on the potential dangers of wild mushrooms as well as removing mushrooms immediately from the reach of pets and kids. I hope that Shiloh’s story will continue to protect others from the potential dangers of deadly mushrooms. 

Tami Mungenast, shilohsstory@gmail.com

September 2008

Shiloh’s Vet, Dr. Carolyn Orr, took the time to write a veterinary perspective on Shiloh’s case. Since Shiloh was never observed to eat any mushrooms, it was a very difficult case to figure out. Read Dr. Orr’s account here.

Fungi on Science Friday!

A dispatch from Kathie Hodge, who is always ready to explore other media

We all know that fungi are cool, and we are all baffled and saddened that they just don’t get enough press. Well that’s all changed now. I was on National Public Radio last week, talking fungus on the venerable show Science Friday. David Fischer joined me to field calls, and there were feature interviews with Kelli Hoover (who discovered a yeast symbiont in the bellies of Asian longhorn beetles), and Arturo Casadevall (coauthor of the hot American Academy of Microbiology colloquium paper, The Fungal Kingdom: Diverse and Essential Roles in Earth’s Ecosystem).

You can listen to the Sept 12 show here.

and earlier in the show, Gavin Sherlock on the hybrid origins of lager yeasts.

Also, check out the Sci Fri video, which features MSA President, Roy Halling, along with a couple of our own timeless time lapse videos:

(having trouble with the video? view it on the Science Friday website)

Fungi in streams: a leaf nightmare

Small fungi with handsome spores attract a select and discerning group of people, among them this anonymous blogger, who is not a leaf but rather a graduate student in Plant Pathology and Plant-Microbe Biology at Cornell.

If I was a leaf, I wouldn’t want to fall into a stream. I’d probably try my hardest to get myself into some kind of a dried flower arrangement, or pressed onto a greeting card as an emblem of autumn. But I would stay away from streams. Streams don’t look like they’d pose much of a danger; after all, they’re just water. But what some leaves don’t realize is that in that water are hundreds of tiny fungi and bacteria just waiting to attack them and break them down, piece by piece, to their simpler elements. And this, for a leaf, must be a very frightening prospect.

A daunting collection of leaf-eating fungi lurks in the waters of freshwater streams. A few are basidiomycetes or oomycetes, but most are ascomycetes and their asexual stages. Stream-dwelling asexual fungi are frequently dubbed Ingoldian hyphomycetes, in honor of the mycologist C.T. Ingold, who first described them in detail. The Ingoldian hyphomycetes include roughly 290 species, including both ascomycetes and basidiomycetes (Shearer et al., n.d.). They are the most commonly studied group of aquatic fungi, and are the main decomposers of hapless leaves that fall into flowing stream waters. You can easily find the spores of Ingoldian fungi by looking in the foam that accumulates in fast-flowing streams–their unique shape (more on that later) means they are easily trapped in bubbles.

Besides the aquatic fungi, streams are also home to some terrestrial fungi that variously find their way there, often by hitching a ride on leaves or other materials that fall in, or perhaps by tossing their spores into the flowing waters. It has been hypothesized that most aquatic fungi evolved from terrestrial fungi whose spores frequently landed in streams. As a result of this evolutionary history, several genera of fungi, such as Ascotaiwania and Annulatascus, include both terrestrial and aquatic species (Wong et al., 1998).

Although they may be scary for leaves, aquatic fungi are looked upon pretty favorably by the other stream dwellers. This is because fungi —especially the Ingoldian hyphomycetes— supply much of the energy in stream ecosystems. Ingoldian hyphomycetes eat the tough lignin and cellulose components of leaves that fall into streams and transfer the energy from the leaves into forms (proteins, fats, phosphate minerals, carbohydrates, etc.) that other freshwater animals can process. These leaves and other organic materials that fall into streams–which collectively are called allochthonous materials–have been estimated to provide 50-99% of the energy in freshwater communities (Barlocher and Kendrick, 1974).

Besides leaves, some aquatic fungi can also degrade tough animal products such as insect exoskeletons, hair, and fish scales. A few are also equipped with specialized enzymes to digest wood that falls into streams (Wong et al., 1998).

If allochthonous materials haven’t been digested by fungi, stream animals have a hard time digesting them and end up pooping out the majority of what they eat. Without being initially processed by fungi, as much as 95% of leaf litter can be pooped out by stream animals, suggesting that they are only able to retain the remaining 5% for use in their bodies (Barlocher and Kendrick, 1974). This small percentage of non-pooped material does not contain sufficient energy to support the necessary biological processes of most freshwater fishes. (Luckily, when this happens there are usually fungi around to digest their poop and make its energy available for the rest of the stream community).

Although “energy flow” sounds like a sleek and elegant process, the reality is a bit woollier. A series of competitive and predatory interactions are involved: When a leaf falls into a stream, spores produced by Ingoldian hyphomycetes float by and their special shapes help them attach to leaf surfaces. Spores of these fungi are commonly tetraradiate–imagine a tripod shape with an extra spike on top, or a jack. Tetraradiate spores can adhere and germinate very quickly (important when you’re tumbling rapidly down a stream). As they attach to the leaf, the spores germinate at the points of contact to attach more firmly. From there they grow into the leaf and begin to eat it.

Autumn leaves are hardly sterile, and typically come with their own collections of terrestrial fungi. These fungi are an early competitive factor for Ingoldian hyphomycetes, but their prevalence drops off as the leaf becomes submerged and its temperature falls–two conditions for which aquatic but not terrestrial fungi have developed adaptations (Wong et al., 1998).

The first Ingoldian hyphomycetes to arrive on a fallen leaf are usually the ones that dominate throughout its digestion. Typically there are four to eight dominant species on any given leaf, and these species change from leaf to leaf. Various rare species come and go, especially during the early stages of digestion (Barlocher, 1982).

Bacteria also enter the leaf community and join in breaking down tough materials. Early scientific research on leaf litter decomposition focused almost exclusively on these bacteria, but recent work suggests that not only are fungi needed to start the initial break-down of leaves, but that fungi are the principle organisms involved in converting leaf materials to a more palatable form (Pascoal and Cã¡ssio, 2004). Bacteria and aquatic fungi compete for available leaf space–fungi are often on the losing side of this battle–but sometimes the bacteria and fungi coexist, each focusing on the types of materials they are best equipped to digest: low molecular weight compounds for bacteria and high molecular weight compounds for fungi (Mille-Lindblom, 2005).

In the later stages of leaf degradation, Ingoldian hyphomycetes are faced with grazers like nematodes, amoebae, and rotifers, which seek out and eat areas of leaves with large amounts of fungal mycelium (Wong et al., 1998). It turns out that not only are these animals attracted to fungal areas on leaves because the leaf is more palatable, but also perhaps because the fungal mycelium provides them with a valuable source of nutrition (Kendrick, 2000).

Don’t worry, fungi often get the last word when predacious fungi turn up to trap and feed on nematodes, amoebae, and rotifers, thus adding greater complexity to the aquatic food web. To wrap it all up, shredding insects like stone fly larvae eat all the aforementioned organisms as well as the digested leaves. The crumbs that they generate wash downstream and are captured by filter feeders like baby caddis flies. And any of these bugs might get eaten by carnivores like fish (Barlocher and Kendrick, 1974). With each step of this food web, energy is transferred from one organism to another, and from one trophic (energy) level of a freshwater community to the next. And so an entire food web is built around a community of fungi and a poor defenseless leaf.

The Dish on Deliquescence in Coprinus Species

This post was written by Jonathan Landsman, who took my Mushrooms course in Fall 07. He is a busy graduate student of Horticulture, here on the Cornell Plantations Public Garden Leadership fellowship.

Good grief, who would have thought that a blog on fungi would be so full of disgusting things? Seems like every fungus is out to make its own kind of mess: molds turn your strawberries to mush, stinkhorns turn your mulch into putrescence, and inky caps (genus Coprinus) turn themselves into a black goo as you see here. I gotta say, I love mushrooms but I wouldn’t want to have to clean up after them.

Quicktime 5+ movie

 

But don’t judge a fungus by its hygiene–the inky caps are some cool ‘shrooms! One species, tippler’s bane (Coprinus atramentarius) contains a chemical that will make you sick if and only if you drink alcohol. Another, shaggy mane (Coprinus comatus) is one you can savor battered and fried, but only if you can detect this delicacy before it deliquesces!¹

To deliquesce is to liquefy. Most mushrooms don’t last more than a few days, but inky caps are usually here and gone in less than 24 hours! They have the fairly rare property of autodigestion, a habit of destroying themselves with their own enzymes once the mushroom has released its single round of spores. I’m a big fan of things that are even more slovenly than I am, so I’ve decided to make the why and how of deliquescence the topic of this blog post.

The Benefits of Digesting One’s Self

Inky caps are not mushrooms you want to carry home in your shirt pocket! The mushrooms appear in wet conditions, but then the same moisture that brought life to the new tissue facilitates death: the inky caps digest themselves via hydrolytic enzymes, tiny biologic machines that use water to break down molecules. The slopfest takes just a few hours, from pileus to puddle. Then the once-mighty Coprinus comatus turns from a mushroom that can split asphalt into a thin liquid. You can actually use this liquid as a semi-permanent ink . . . it’s only a matter of time before the practical joke industry discovers inky caps.

But why would a living organism destroy part of itself? One reason is a bit like why the husk of a milkweed fruit becomes brittle and cracks once the feathery seeds within are mature. It’s a selective-death process that get useless tissue out of the way of the babies, letting them escape to as far from the parent as possible before they begin their new lives (it’s a little like college). As an inky cap deliquesces, the tightly packed gills separate and curl back, allowing some spores to float out into the air.

Self-sacrifice by the parent is not unknown in the animal world either (as those paying for college know). Ever heard of how praying mantis females eat the males after mating? Some entomologists believe that he sacrifices himself to provide additional nutrients to the mother and her eggs, and C. lagopus and C. curius are actually similar. These inky caps (and probably other species too) transport all of their sugars out of the fruiting body and into their spores just before sending them off. By the time deliquescence begins, there’s virtually no food left in the cap or gills; it’s all gone to the spores.²

The Disgusting Details of Deliquescence

If it’s worth it to you to get a little technical to learn even more about how mushrooms turn to muck, this section is for you.

We already mentioned that hydrolytic enzymes are responsible for breaking an inky cap down into goo (“goo” is a technical term). Hydrolytic enzymes are not unusual; fungi secrete them to digest complex organic molecules into a nutrient slurry that their root-like mycelia can drink in.

What makes inky caps special is how they make and use another kind of hydrolytic enzyme. It’s chitinase, a group of enzymes that break down chitin, which is a complex sugar in fungal cell walls that makes them sturdy. Chitinase is not found in the fungus at all until just after meiosis has produced spores. At this point, about two hours before spore-release, chitinase is made, but only in the mushroom cap and gills, never in the mycelia or stem.³

Since chitin is present in all the cell walls of a fungus, you can imagine what chitinase, the chitin destroyer, does to an inky cap once it gets going. Deliquescence is a chain reaction, starting with the end of the gills closest to the mushroom stem. Spores are released from here first, and then autodiestion of gill cells there takes place, releasing a liquid that is a potent digestive. The liquid is taken up by the neighboring cells, which are turned into more liquid, and the wave of destruction travels along the gills just behind the region of maturing spores. Thus the mushroom is digested from the center to its fringe.⁴

If you were intently worried about the future of the Coprinus race, this story might raise a few concerns for you. If chitinase is sweeping down the gills at such a rate, why isn’t it destroying the spores before they can be released? It may be that many spores are caught in the liquid and perhaps destroyed (though the literature is silent on this point), but the fact that enough are dispersed to perpetuate the species might have something to do with the unusually high degree of coordination in spore development in at least some species of inky caps [see Editor’s note below]. In Coprinus cinereus, 60 to 85% of all the basidia (spore-bearing cells) in one cap will be in the same stage of development. That requires a remarkable deal of communication, and researchers suspect the coordination utilizes light signals: it is known that the mushrooms require light to develop normally.⁵

Deliquescence in Poetry

Here’s something totally weird: even though chitinase digests chitin, and chitin is present in the walls of all fungal cells, Coprinus chitinases don’t seem to be effective on the cell walls of the mycelia or stipe. When the mushroom cap melts, these parts remain, as you can see in the picture at left.^{4, 5}

This mystery has given us at least one work of art. Percy Bysshe Shelley is believed to have been inspired by an inky cap when he wrote

Their mass rotted off them flake by flake

Til the thick stalk stuck like a murderer’s take,

Where rags of loose flesh yet tremble on high

Infecting the winds that wander by.

   –”from “The Sensitive Plant,” Percy Bysshe Shelley, 1820⁶

See how the “thick stalk” is left behind? Only the gills and cap are digested; their ragged remains and the untouched stipe are left behind in Shelley’s vision.⁵

So you want to be a truffle-farmer…? (Part 2)

This post and the one before were written by the same student in my Fungi class in Fall 07. No, she hasn’t moved the France (yet).

In part one, you traveled to France to pursue your dream of establishing a truffle farm. After several months traveling the country, you have realized that truffle-farming, especially in Southern France, is serious business, and you decide your entrée into the truffle industry would be greatly aided by a better understanding of the history of this distinguished fungus.

As an integral part of French economy and cuisine, the truffle is firmly entrenched in French culture and society. A source of national pride, the “Black Pearl of Pé rigord” (T. melanosporum) is the variety chiefly cultivated throughout France (8).

Although French attempts at truffle cultivation date back to the Renaissance, successful and commercial cultivation of the Black Pearl of Pé rigord did not arise till the early 1800s. Joseph Talon, a French peasant, receives credit for the first active cultivation of truffles when in 1810, this industrious fellow planted some acorns on his small plot of land. He found, several years later, that truffles had grown in the organic soil layer around his young oak trees. A shrewd and enterprising young man, Talon began purchasing “worthless” land on the cheap, and planting it with seedlings grown under the original 1810 trees. Over the years, he amassed a veritable plantation of oak trees and truffles, and was able to make a profit of his thrifty investment. Talon divulged his knowledge of trufficulture later in his life, and his cousin, a man by the name of Rousseau, is credited with sending the first lot of cultivated truffles to Paris for sale in 1855. The arrival of truffles for sale in commercial quantities, along with Rousseau’s publicity of Talon’s methods for truffle-growing, sparked a flurry of interest and research as curious minds promoted truffle-cultivation as an industry (8).

Today, growers often still use acorns and oak (or hazelnut) seedlings to establish truffle plantations. After 3-5 years of cultivation, you should begin to see bare regions around the trees where grass has died due to fungal growth in the soil (6,8). This phenomenon, the brûlé , is probably a result of allelopathic compounds secreted by the fungus. At this point, further decisions on plantation management must be made. Two methods of cultivation have emerged over the years, which capture the two extremes of modern truffle management.

The Tanguy method is often favored by those with smaller budgets and who prefer a less-intensive management strategy more closely resembling truffle growing under wild conditions. Growers use mowing for weed control, but eschew other management strategies such as fertilization, soil aeration via plowing, and intensive irrigation. Truffles generally take a few years longer to produce a harvest under these conditions, but the approach limits drastic alteration of the original environment (6).

The Pallier method is more management-intensive, as farmers control weeds and promote soil aeration by tilling or harrowing in the early spring and summer. Trees are also pruned to allow maximum sunlight to reach the soil and the developing fungi, and irrigation maintains adequate soil moisture levels. While such strategies may promote higher yields and give the grower more control of the environment, a risk remains of damaging roots and soil with tilling and excess irrigation. This could lead to reduced truffle yield and might support the invasion of competitive mycorrhizal fungi (6).

Depending on your choice of management, you can begin to look for truffles after 7-15 years of cultivation. For Périgord black truffles, harvest occurs from mid/late winter through May. The annual arrival of truffles in French markets is much-anticipated and widely-publicized at famous markets such as those at Richerenches in Provence, said to be the largest in Europe (1).

Truffle production in France reached its peak near the end of the 19th century, with a reported yield of ~2200 tons of fresh truffles in 1890 (3). Political disorder and geographical disruption resulting from World Wars I and II caused a sharp decline in truffle yields. While the industry has regained its solid footing in the following decades, current production levels are still far below their peak at the turn of the last century (4).

Today, truffle cultivators and those who harvest from the wild face a new threat in the form of global warming. You discover that even these fungi, for all their laudable qualities, are not immune to climate change. Because of droughts in France during the past five years, truffle production has dropped significantly. In 2005, France reported only ~37 tons of truffles, much less than previous decades (5).

Tuber melanosporum also faces competition in the form of several Chinese truffle species, most notably T. sinensis and T. himalayensis. These two species strongly resemble the color and shape of the Black Pearl of Périgord, so much so that it is difficult to distinguish between species without a trained eye (2).

In light of shortages and rising prices, an even more blatant impostor has emerged in the form of truffle oil. Mounting a challenge to T. melanosporum‘s share of the market, this oil does not even usually contain any truffles; rather, it receives its pungent odor from a chemical compound (primarily the sulfide 2,4-dithiapentane). While many chefs and food critics decry this oil as an inferior substitute for the real truffle (which has a more complex odor profile), the laws of supply and demand appear to overrule these purists’ objections, and the relatively inexpensive oil has carved a niche of its own (7).

Your time in France has increased both your knowledge of the truffle industry and its place in French culture and society, as well as your fascination with this fungus. Despite the current challenges, you feel that the persistence of the truffle in its purest form remains an integral part of French society, and believe the market for truffles will persist even in the face of current challenges.

So you want to be a truffle-farmer…? (Part 1)

This post and the next were written by a student in my Fungi class in Fall 07. We ate some truffled cheese together and I guess she liked it.

Perhaps you are facing a mid-life crisis. Maybe your current occupation is dismally unfulfilling. Or mayhap you are a soon-to-be graduate of a local university and have no post-graduation plans, much to your parents’ dismay. Regardless of your age, if you are dissatisfied with your current position in life, you might consider a career as a truffle farmer. What is a truffle-farmer? and, Why would you ever want to be one? These are questions you will doubtless be asked by inquisitive friends and concerned family members, once you announce your decision to farm truffles for a living. In order to quiet their fears and prove that your fascination with these fungi is a legitimate motivation for a career change, you will need a little bit of background information.

First, you will probably need to explain what truffles are, in the mycological sense. After establishing the fact that you are not pursuing a career in chocolate-making, you explain that truffles are the fruiting bodies of a particular group of fungi in the phylum Ascomycota. The order Pezizales, which also includes delicious morel mushrooms, houses the family Tuberaceae, itself home to a range of truffle species (3,4).

You give your family and friends some basic truffle morphology, explaining that these particular fruiting bodies (called ascomata) closely resemble clods of dirt, with their irregular spherical shapes and dark, often bumpy exteriors. You mention that the interior of truffles consists of densely-packed reproductive tissue, with white “veins” throughout, and studded with beautiful ascospores. You share some truffle trivia, stating that, unlike most ascomycetes, these fungi do not actively release their spores, and grow hypogeously (under the ground) (3,4). You show them a photo of a freshly harvested black truffle, and a cross-section revealing the dense, marbled interior.

When you are further questioned about your affinity for these fungi, you point to their long culinary lineage, noting that truffles were regarded as delicacies by the ancient Greeks and Romans, and that their culinary worth persists to the present day (4). You affirm their economic value by stating that truffles are often sold for hundreds or even thousands of dollars per pound. You assert the ecological benefits of these fungi, describing how they live in mycorrhizal association with the roots of several tree species, particularly oaks and hazelnuts, providing more surface area for nutrient uptake by the tree roots (5).

After laying down a convincing argument for the legitimacy of your career path, you decide to put words to action. You realize, however, that there are a number of things to consider before you can launch your career as a truffle-farmer.

While there are many varieties of truffles used for both cooking and canning, including the Italian white truffle, the Chinese truffle, and even the desert truffle of the Middle East, you disregard them all in favor of the titan of truffles, Tuber melanosporum (2,5). Known commonly as theBlack Périgord truffle for its French place of origin, this esteemed fungus is the most highly prized and economically valuable variety on the market, often selling for over $1000 per pound (6).

As T. melanosporum is native to France, you conclude that the best place to begin your career is in the southwest countryside of France, where the distinct seasons and the temperate climate favor truffle growth (2,5). Well-meaning relatives point out that truffle cultivation is not exclusive to France, and suggest you choose someplace else such as the northwestern United States, Italy, New Zealand, China, Australia, Tasmania, or even the Middle East as an alternative (2). You reply that you prefer France, as it is the best place to enjoy truffles of the fungal and chocolate variety.

When friends and family point out that they don’t know a lot of truffle-farmers, and suggest that you might be lonely in your new profession, you allay their fears by explaining that you will have a constant companion in your hand-picked truffle-hog. An essential to truffle-farming, the truffle-hog (or truffle-dog, should you prefer canines to swine), would be used during truffle-harvesting season (usually from November to March) (6) to detect the strong, earthy odor of the truffles in the soil. As truffles mature underground, you explain, they emit an odor which attracts pigs, and can be recognized by dogs as well (2,3).

After choosing a variety, a region, and a truffling companion, you realize all you are lacking are the truffles. Doing some research, you discover that there are businesses which specialize in the production of trees with roots already inoculated with T. melanosporum (5).

You make plans to purchase some trees, but your well-laid plans are almost derailed by an unanticipated bout of sound reasoning. You realize that truffle farms take some years to come into their full glory, as the fungi and the roots must develop and mature before any significant harvesting is possible (5). Refusing to give up on your dream, however, you decide to travel to France and apprentice yourself to an established truffle farmer, to learn the tricks of the trade and establish your trees. You astutely pick up a copy of the new book Taming the Truffle as your bible (7). Then you depart for France, confident in your new life direction, and promise to write family and friends of your experiences in the French truffle industry.

continued in part two…

The Future of Fungal Freshness?

This post was written by a student, who we’ll affectionately call student12. He wisely took two of my classes in Fall 2007, Fungi (PLPA 309), and Mushrooms of Field and Forest (PLPA 319)

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Do you like fresh food? That’s a silly question, right–who wants to buy limp, old looking food? No one! That is why supermarkets have entire sections devoted to “fresh produce.” But what do they mean by “fresh?” How can produce that was harvested a thousand miles away and a week or two ago be “fresh?” That doesn’t seem to fresh to me! How would you feel if you knew your food was truly fresh? Better yet, how would you feel if your food was fresh that day? And even better yet, how would you feel if you knew your mushrooms were fresh that day? Sounds too good to be true! But it’s not–thanks to Agata Jaworska. Agata may have changed the face of fresh with her Masters thesis concept, ‘Made in Transit.’ ‘Made in Transit’ is a supply chain concept in which the food grows on board a vehicle on the way to the supermarket, shifting the paradigm of packaging from preserving freshness to enabling growth, and shifting ‘best before’ to ‘ready by.’

I love eating mushrooms, and I can’t tell you how many times I have gone to the supermarket with mushrooms on my mind, only to be sadly disappointed by a not-so-fresh looking display. Once I heard about Agata’s concept, I had to know more. I contacted Agata and asked her for an interview. Here is what she had to say:

Q: Can you give us a brief update on what is going on in the area of “ready by?”

Agata: In a way, the concept of adding value on the way to the market exists. For example, though most of the time bananas are ripened in separate land-based ripening rooms after they arrive, companies are starting to ripen bananas on board the shipping containers, by building the conditioning capacity of ripening rooms into the shipping containers. So, though bananas are not “made” on the way, they are starting to be ripened on the way.

But in chain cost calculations, transport, as far as I know, is always considered a cost factor and never a value-adding factor. I don’t know of an instance in which on-the-way production is a dominant part of the production plan.

Something related to the concept are potted plants (take potted basil for example), which the consumer takes home still in the pot. The distinction is that basil is not grown in transit, but rather it is kept alive. In fact, the growth of let’s say chives is prevented while in transit, because it wouldn’t be the right sort of growth (because conditions are not optimized), so even potted herbs are preserved on the way, and not grown on the way.

Q: What are you currently doing in the area of “ready by”? What is the nature of the project(s) you are currently working on? Who are you currently working with?

Agata: Since graduating from Design Academy Eindhoven in June, the concept has been exhibited at various design events (currently it can be seen at an exhibition focused on sustainability, “Living and Working Together” in Utrecht, The Netherlands, and next it will be part of “Design and the Elastic Mind” at MoMA in New York), I have also been presenting it to industry (fresh food & packaging) in cooperation with the Netherlands Packaging Centre. Through this exposure we are gathering interest to launch the next level of (predominantly scientific) research that needs to happen, and the industry collaboration. I am working with the Systems & Control group, and also the Mushroom Group at Wageningen University to put together a proposal for further funding. We will be looking for PhD students as well (we’ve decided I may not be the best mushroom researcher after all!).

Q: What species of mushrooms (have you, can be, do you envision being) grown using “Made in Transit” methods? What are the major considerations when choosing mushroom varieties to grow?

Agata: We have done some very preliminary testing with white button mushrooms and grey oysters, but not in a way that cultivation methods are designed around the “Made in Transit” concept, but rather adapted more or less standard ways of growing these species and sent them on their way. It would get a lot more interesting when we start to design the way of cultivating around the reality of transport, much more than is done today.

The kinds of species that would most benefit from the concept are the kind that are extremely fragile (and therefore currently cannot be transported because they begin to liquefy when subjected to refrigeration or vibration), and the kind that have a distributed customership (so therefore it doesn’t make economic sense to set up a local farm). Paddy rice straw mushroom is one such example.

Q: What other foods can be (can potentially be) grown using “Made in Transit” methods?

Agata: We could bake bread on the way to the market, in a sort of mobile bakery delivering the freshest goods. Baking can happen on board on the way, which could delay the order and make the chain much more flexible and responsive. Micro vegetables are also a possibility. But I’m sure there are others.

Q: Do you expect to obtain consistent yields and appearance? How does the package design impact produce yield and produce appearance?

Agata: The package I designed (and which was produced by Voges Packaging) is based on the way oyster mushrooms are cultivated now, only adapted to an individual level. Readers of this blog will also notice that what I call the “growth pad” is too small in proportion to the size of fruit bodies. This package is futuristic in the sense that the substrate is smaller than what is currently possible. With the group at Wageningen University we hope to work on this, amongst other things. This concept could only be realized once we are able to control yields and appearance. As long as we’re using the same terminology, I’ve seen the difference in appearance of the sporeless variety of oysters developed by the group in Wageningen, so I’m not too worried about that. The package will have to be designed to create the most ideal condition for growth as possible, and in principle, it should be easier to control the outcome when you are working with an individualized container than on a bulk level (i.e. a package and not a whole room). The conditions surrounding the package will also be controlled.

Q: What are some of the greatest technical difficulties of growing mushrooms in transit?

Agata: The greatest challenge is that realizing this concept involves a lot of change on a system level. It’s kind of a revolution and not an evolution, precisely because it is a paradigm shift. But, I don’t think it’s that far fetched since we already have some of the capacity on board, we just don’t put it to this use. Containers already have the capacity to control environmental factors (humidity, temperature, carbon dioxide levels), they just are not typically set to enable growth, but rather to preserve freshness. A lot of the technology exists already, but it just means that we have to build it into the system, which is kind of a big change.

Q: On average, how long does it take for a package to grow? On average, how long will it take to arrive? What mechanisms are available to slow growth in the case when arrival time is expected to be much longer than grow time?

Agata: Remember we are still in the theoretical realm (as this is a futuristic concept) but I’d say 5-7 days. It would be more or less the same as current fruitification times. It is also possible that packages arrive prematurely to the supermarket and the last part of growth could happen there. It would be like bananas that you can buy green or yellow, depending on how soon you want to eat them. Just check the “ready by” date and pick the one you want.

In terms of mechanisms to control growth, it would involve controlling the environmental condition in the vehicle (temperature, etc.), but this and more is yet to be developed.

Q: Given that mushroom harvesting can account for up to 40% of the cost of production using traditional methods, what is the cost breakdown using the “Made in Transit” paradigm?

Agata: Good question, we’re not there yet. Harvest cost is zero (and note that harvest labour no longer exists, since for the consumer picking his own mushrooms prior to cooking is more fun than work, I think), on-site production cost would go down (we wouldn’t need growing rooms, just packaging facilities), packaging cost would go up because the substrate would be built into the package (and we hope to make the protein-enriched substrate edible) and ideally transport costs would stay the same. Though people may be willing to spend more money on the substantial jump in quality, I am more interested in testing the economic viability of this new mode of production. Of course it may not be such a good idea to start with white button mushrooms since there is such a low margin to begin off with.

Q: What impact do you feel the “Made in Transit” paradigm will have on movements to buy locally grown foods and to support local agriculture?

Agata: Developments in local agriculture can go on as normal, just as developments in my mother’s garden will also go on as normal. For this project I was interested in tackling global chains and wondered if they could be done differently, and indeed address their sustainability. For the next project, I may tackle local food production.

Q: What is the general market acceptance of such a high-tech grow method?

Agata: Indeed, next time a kid asks me where mushrooms come from, I’ll have to tell him that they may soon (or not so soon, depending on our research funding and industry partners) come from trucks! And is this a utopia or a dystopia? Well it’s not as romantic as going to the forest but I hope it turns out to be more sustainable than the way it is currently done, given our global state of affairs. I think it shows that sustainability is not as clear cut as one would think, and dare I say, that local is not always better than global?

Q: When will “Made in Transit” products be available to the public?

Agata: No idea. It really depends on when we can start the research and find industry partners that want to work with us.

Q: What is your vision of “Made in Transit” for the future?

Agata: I would like to find more applications and develop new business models. I most look forward to working with people from different fields. With this project I already have experienced this, and it was one of the more gratifying things. When you are sitting around the table with professionals and researchers and you don’t have to say a thing because the enthusiasm is already there. It’s nice how such a concept can do that.

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Agata Jaworska graduated from Design Academy Eindhoven with a Master of Design in 2007. She is currently looking for scientific researchers, industry partners and collaborators to make the concept a reality. She can be contacted at jaworska.mail@gmail.com.

If you would like to learn a little more about Agata’s design concept “Made in Transit”, check out this two-minute animation (Agata says “The guy is shopping, picks up mushrooms with a ‘ready by’ instead of ‘best before’ expiry date, and then we go back in time to see how the mushrooms are made and find out that the chain starts in the packaging factory and the mushrooms are grown in their package on the way to the supermarket.“):

Youtube Two minute animation (or view the Five minute presentation)

Student12 says: I would like to thank Agata for taking time to correspond with me about her fascinating Master’s thesis concept. And I look forward to the day when truly fresh mushrooms will be available at every grocery store.

A simple way to preserve fungal cultures

This post was generously written by Anuar Morales, a doctoral student in department of Entomology at Cornell University. He works on insect biocontrol.

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My field, biological control, is served by several culture collections of fungi, bacteria, viruses and nematodes (collated at WDCC). In these collections it is possible to find a natural enemy of just about any insect pest. Collections also support conservation of earth’s biodiversity in the face of globalization, habitat loss, and climate change–pressures that threaten microbial species as much as whales and tigers.

Culture collections are expensive to support, as they require special equipment and continuous attention in order to maintain fungal cultures without losing their pathogenicity or virulence. Two examples of these collections are the USDA-ARS Collection of Entomopathogenic Fungal Cultures, with more than 5500 cultures of over 350 species of fungi from 900 hosts. The International Entomopathogenic Bacillus Centre in the Institute Pasteur has nearly 3500 strains of Bacillus thuringiensis, the most important bacterium used in biocontrol. Cultures are typically either freeze-dried in a process called lyophilization, or stored in liquid nitrogen at ultra-low temperatures. Both techniques require intense labor and expensive equipment.

At the International Center of Tropical Agriculture (CIAT, in Colombia), expenses were reduced with a novel, reliable and cheap technique. The dry filter paper technique was developed by Rosalba Tobon and Ximena Aricapa in the early 1980s and can be used for preservation of cultures of insect pathogenic and plant pathogenic fungi as well as many molds.

The first step is to isolate the fungus into pure culture. A tiny pinch of the fungus is taken directly from the insect or plant host and added to a Petri dish containing culture media. If the identity of the fungus is known, selective culture media might be available; if not, general media such as PDA (Potato Dextrose Agar) can be used for the initial isolation. Lactic Acid or Chloramphenicol can be added to any standard medium in order to reduce contamination by bacteria. After a pure culture has been obtained, the fungus is grown for 5 to 10 days. Now the storage process can be initiated.

In the second step, pieces of filter paper of about one square centimeter are cut and sterilized in an autoclave. They are then placed on the agar surface of the same selective or general medium used to isolate the fungus.

Fresh spores or a little piece of the pure culture is cut from a fresh colony, and placed on top of each piece of filter paper (be sure to work in a sterile environment). The Petri dishes are sealed and placed in an incubator–the fungus typically grows more slowly on filter paper, needing approximately 10 to 15 days to fully colonize it.

Once the fungus begins to sporulate on its filter paper, the individual pieces of paper bearing fungus are separated from each other and the underlying medium, then placed in new Petri dishes without any culture medium. After that the Petri dishes are put again into the incubator until the paper and fungus are completely dried, approximately 20 to 30 days.

The drying process is most crucial because if it is too fast, the fungus can lose pathogenicity and virulence or be killed; and if it is too slow it can become contaminated by other fungi or bacteria.

As soon as the fungus is dried, 10 to 12 pieces of paper filter are put in a sterile glassine envelope. Each envelope is labeled, bagged in a plastic bag and it stored in a plastic container at 4°C or -20°C depending on the facilities that are available. When a fresh culture is needed, one small piece of filter paper is removed from the envelope and placed on fresh medium.

The new technique is not only reliable, it is very inexpensive and easy to use in any laboratory with few resources. CIAT uses this method to store about 500 cultures of insect pathogenic fungi and 1000 cultures of plant pathogenic fungi and bacteria. Evaluations of purity, pathogenicity and virulence were performed on fungi stored between 5 to 10 years. With a few exceptions the fungus was recovered easily and with the same characteristics of pathogenicity and virulence it had when first stored. This technique has been successfully implemented in other institutions with great results. Research studies at CIAT are adapting this methodology to work with bacteria and viruses.

The Perfect Pitch

It’s nice to have guests once in a while, especially around the holidays. Today we welcome Elio Schaechter and Merry Youle. They first wrote and posted this piece on the exquisite blog, Small Things Considered. The story is too good to miss; it is duplicated here with permission.

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A spore print of an Agaricus.

Source: GL Barron, U. of Guelph

If you’re not into mushrooms, you don’t know what you’re missing. (Find out what I mean by having a look at my book on the subject.) When hunting for wild mushrooms, all your senses will be satisfied, and so will your scientific curiosity. Consider the dispersal of fungal spores and the variety of ways that fungi have evolved to do it. Some use wind transport, others employ water droplets, yet others induce insects to carry their spores for them. An amazing world. Here we will contemplate how mushrooms do it.

As a kid, you may have made a spore print. For those who haven’t and to remind those who did, a spore print is what you get when you take a mature mushroom, cut off the stem, and place the cap (gill side down) on a piece of paper or plastic. If all goes according to plan, after an hour or so, you’ll see the spores deposited on the paper in a pattern that corresponds to the spaces between the lamellae or gills on the underside of the cap.

Mechanism of Spore Discharge. The bars are 10um. A. Images

taken at 10 sec intervals with a conventional camera. Credit:

John Webster. B. Images taken at 10 μsec intervals with ultra

high speed video. Credit: A. Pringle et al. The captured launch

of a ballistospore (2005) Mycologia 97(4) 866-871. Courtesy

of Nik Money

How did this happen? There is magic here. Since the gills are usually moist, the spores have to clear the gill surface to avoid getting stuck there. The mechanism for carrying out this feat has been further elucidated in recent years by Nik Money and colleagues at Miami University in Ohio and Anne Pringle, now at Harvard. One of their tricks was to use an ultra high speed video camera, one capable of upwards of 200,000 frames per second. This permits investigators to observe events that happen at very high speeds. The discharge from the gill surface proceeds at an initial speed of some 1500 μ/msec and at an acceleration greater than 10,000 times the force of gravity, which is impressive. But the spores have a problem. If they were to travel too far, they would hit the surface of the adjacent gill and nothing would be gained in the process. Actually, spores pop from their mother gill fast but they do not go far. After traveling about 0.1 mm, they abruptly lose momentum and fall straight down. Outdoing baseballs thrown by the best of pitchers, they change direction suddenly from a horizontal trajectory to a vertical one-the ultimate in curve balls! A nice account can be found on the Website of the Australian National Botanic Gardens.

So, how do these spores (also known as “ballistospores”) get ejected? Here we’re talking about regular mushrooms, because other kinds of fungi have their own mechanisms. In mushrooms, the spores are made after meiosis by budding off a specialized surface cell called a basidium (hence, Basidiomycetes). Each spore is held in place by a thin peduncle (the sterigma) that breaks to release the spore into space. If you imagined that the force involved is osmotic turgor or gas pressure, you’d have guessed wrong (although this is the case for some other fungi).

To visit the actual mechanism, we must recount the pioneering work from the early 1900’s by Reginald Buller, a Briton transplanted to Winnipeg. (An aside: It is said that in order to keep his eyes adjusted to dim light during work on bioluminescent fungi, he walked along the streets from the hotel where he lived to his lab wearing horse blinders.) Buller noticed that, immediately before discharge, a water droplet formed at the base of the spore. The drop is formed by the secretion of mannitol and other osmolytes into the moist environment of the gills. What Money and collaborators observed with their ultra high speed camera was that this “Buller’s drop” grows until it abruptly coalesces with the water on the surface of the spore. This shifts the spore’s center of mass towards the gill, which is sufficient to detach the spore and release it into space. (Image 3 around here) In Money’s words: Fluid movement is powered by the reduction in free energy (surface tension) when the two drops fuse, so we refer to the mechanism as a surface tension catapult. I have been trying to find something analogous to this in everyday experience but have failed so far. Sticking to the baseball analogy and pretending that the spore is hit by a watery bat doesn’t work. Readers are welcome to contribute their own imagery.

A Diagram of the Mechanism of Spore Discharge. (1 & 2) At its base, a spore secretes a small amount of sugar, which absorbs water, thus forming the Buller drop. The growing drop causes the center of mass (red dot) to shift as shown by the red arrow. (3) At the same time, moisture is also condensing on the spore surface (blue). (4) When the drop comes in contact with this surface film, it collapses, shifting the center of mass in the direction of the arrow and launching the spore. Source: Australian National Botanic Gardens, Fungi Website. Written by Heino Lepp, updated on web 25 June, 2007 by Murray Fagg.

Sporobolomyces roseus

You may ask, why are mushrooms included in a blog on “small things?” Besides declaring them to be honorary microbes, mushrooms are the fruiting bodies of an organism that consists of microscopic filaments, just like molds. These, of course, qualify as microbes. But suppose you are very picky and are not happy with such a generous inclusion. Well, will you agree that yeasts are microorganisms? If so, we present to you Sporobolomyces, a basidiomycete yeast that is one of many capable of discharging spores by a mechanism analogous to that in the mushrooms. A video from the Money lab captures this process. Although the video was taken at 210,000 frames/s, you must pay close attention to see the spore fly rapidly through space. These spores are not the product of meiosis but are conidia, the vegetative spores characteristic of molds. But they qualify as “ballistospores” just the same as the basidiospores. Isolating these yeasts is very easy. Spread some soil on an agar plate, invert it above another agar plate, and incubate for a few days. The bottom plate will then likely have pink colonies of these yeasts. (Sporobolomyces was first described in 1924 by two of the fathers of microbial ecology, Albert Kluyver and Cornelis van Niel.)

How mushrooms disperse their spores likely has you intrigued by now. But take it from me, this is only one of the many phenomena that will grab your attention, should you be so lucky as to get involved in any of the many facets of mycology.

Our thanks to Nik Money for the movie and for generously sharing his wisdom and experience. Nik has authored several informative and entertaining popular books on fungi, viz. Mr. Bloomfield’s Orchard: The Mysterious World of Mushrooms, Molds, and Mycologists, Carpet Monsters and Killer Spores: A Natural History of Toxic Mold, and The Triumph of the Fungi: A Rotten History.

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