Feeding the Good Bacteria Cut Pepper Root Rot to 6%
Se-Chul Chun, Konkuk University, Department of Molecular Biotechnology, College of Life and Environmental Sciences (professor; plant pathologist) · South Korea
A Konkuk plant pathologist, new to natural farming, reviews biological control of soil-borne disease and shows his own work: bacteria coated on rice seed against seedling rot, and bacteria against Phytophthora root rot of pepper, both working better when given a sugar the bacteria can use and the pathogen cannot. He then gives first, unreplicated microbe counts from the Paju natural farming soils and from each IMO step: counts fell about tenfold from IMO1 to IMO4 and the fungi shifted from Aspergillus to Rhizopus. He draws no conclusion about natural farming yet.
What they found
How do beneficial microbes control soil-borne plant disease, and what microbes are in natural farming soil and in IMO?
Two parts. (1) His earlier biocontrol work, dates and sites mostly not stated: rice seed (water-seeded) inoculated with a Pythium seedling pathogen and coated with antagonistic bacterial strains (D-series, e.g. D324), with and without 8% carbon sources in a talc-based seed coating; a field emergence test; pepper inoculated with Phytophthora capsici and treated with antagonistic bacterium KJIR5 plus 2% carbon sources; root colonization of rice by GFP-tagged Bacillus subtilis CB-R05 in hydroponic culture. Seedling and disease tables give means ± SEM; the rice strain table cites Dunnett's test at P=0.05. (2) New in 2016: plate counts (CFU per gram) of bacteria and fungi in Paju test-farm soils (natural farming, conventional, organic and a control soil, from the peanut trial) and in IMO1–IMO4 made by his lab (IMO1 from forest compost on cooked rice; IMO2 IMO1 + sugar; IMO3 IMO2 + rice bran; IMO4 IMO3 + soil 1:1). IMO1 was kept about two weeks in a refrigerator before the next step. Replication, sampling dates and statistics for the counts: not stated.
- Rice: bacterial seed treatment — Nine antagonistic bacterial strains (D324, D316, D311, D315, D305, D302, D320, D309, D318) on rice seed sown with the Pythium pathogen; compared with pathogen alone and with no pathogen.
- Rice: bacteria plus carbon source — Talc + seed base, with or without D324, plus 8% alanine, galactose, sorbitol, mannitol, glucose or proline; pathogen (Pythium) added.
- Pepper: KJIR5 plus carbon source — Phytophthora capsici alone, plus bacterium KJIR5, or KJIR5 with 2% arabinose, glucose, maltose or melezitose; non-inoculated control.
- Soil counts — Bacteria and fungi in natural farming, conventional, organic and control soils from the Paju farm.
- IMO counts — Bacteria and fungi in IMO1, IMO2, IMO3 and IMO4.
-
Pepper root rot: 81% with the pathogen alone, 22% with bacterium KJIR5, 6% with KJIR5 plus arabinose
Disease incidence (%) ± SEM. KJIR5 + glucose also 6%, + maltose 13%, + melezitose 22%; non-inoculated 0%. A carbon-use chart shows KJIR5 using arabinose well and P. capsici using it poorly.
▶ 13:38 Said and shown on the slides
-
Rice seedling emergence rose from 63% (pathogen, no bacteria) to 80% with strain D324
Non-inoculated seed 92%. Seven of nine strains (74–80%) differed significantly from no bacteria (Dunnett, P=0.05); D309 and D318 (69%) did not.
▶ 11:00 From the slides
-
D324 plus a carbon source the fungus cannot use lifted emergence from 36% to 69–74%
Seed coating with Pythium: base alone 36%, base + D324 44%, D324 + 8% alanine 74%, + galactose 73%, + sorbitol 69%, + mannitol 68%; non-inoculated seed 68%. D324 + glucose (57%) or proline (55%), which the fungus uses well, did less.
▶ 12:19 Said and shown on the slides
-
In the field, sorbitol 8% + D324 gave the most emerged seedlings
Bar chart, values read approximately from a skewed photo: sorbitol + D324 about 460, galactose or mannitol + D324 and base + D324 about 410–425, base about 345, the fungicide Ridomil about 270, untreated seed about 250. Units not stated.
▶ 12:37 From the slides
-
GFP-tagged Bacillus subtilis CB-R05 colonized rice roots within 24 hours
Microscope images show the green-tagged bacteria inside the roots; he says they persisted with little loss. A slide notes images at 30 days after transplanting in hydroponic culture.
▶ 14:18 Said and shown on the slides
-
First soil test: the control soil had the most microbes; natural farming soil had fewer
CFU ×10^5 per g soil, bacteria/fungi: control soil 66.9/10.0, natural 6.3/5.3, conventional 9.1/not detected, organic 14.9/not detected. He says his students' and Professor Kim's students' replicates gave different results.
▶ 17:43 Said and shown on the slides
-
Second soil test, fungi only: natural farming soil 18, organic 13, control 11, conventional 7 (×10^2 CFU/g)
He says the differences may not be statistically significant.
▶ 18:59 Said and shown on the slides
-
Microbe counts fell about tenfold from IMO1 to IMO4
CFU ×10^7, bacteria/fungi: IMO1 151/56.5, IMO2 74/34.5, IMO3 98.5/9.62, IMO4 11.4/4.62. He links the IMO4 drop to mixing IMO3 1:1 with soil.
▶ 19:59 Said and shown on the slides
-
The fungi shifted from Aspergillus in IMO1 to Rhizopus in IMO4
Summary slide: from more sporulating fungi (Aspergillus) to more runner-hyphae fungi (Rhizopus). Species identification of the bacteria not yet done.
▶ 21:45 Said and shown on the slides
- The natural farming part is preliminary and his own words are 'I cannot make conclusion yet'. Soil counts come from two runs that did not agree; no replication or statistics were shown for them.
- Plate counts measure only microbes that grow on lab media, and say nothing of which ones or what they do.
- The control soil in the first test is not described; its high counts are unexplained.
- The IMO was not made in the field way: IMO1 sat about two weeks in a refrigerator before IMO2, and the counts are from one batch.
- The rice and pepper biocontrol work is separate research with lab bacterial strains, not natural farming solutions; sites, years and plot sizes are mostly not stated. The pepper field photo is credited to another researcher ('Adapted (K.D. Kim's)').
- Field emergence values are read approximately from a skewed photo of a bar chart; units not given.
- Glucose, which the pepper pathogen uses well, gave the same 6% disease as arabinose, so the 'feed the bacteria, not the pathogen' explanation does not fit every result.
- Soils can become disease-suppressive: in the US Northwest, take-all of wheat faded after years of continuous wheat because of Pseudomonas bacteria in the soil.
- Beneficial bacteria protected rice seedlings and pepper roots better when they were given a food they use and the pathogen does not.
- In his test, microbe numbers dropped at each IMO step while the kinds of fungi changed; more microbes is not the measure of a good IMO, and he is still studying what the change means.
- IMO comes from the local forest and costs nothing; commercial biocontrol products cost money.
Next to Master Cho's teaching
-
Agrees
Disease as a broken microbial balance Cho: disease is not one bacterium's wrongdoing but a deteriorated relationship between crops and microorganisms, and IMO restores the balance. Chun's disease-suppressive soils and antagonist bacteria describe the same idea in plant pathology terms.
Cho, Janong Natural Farming (2003), p. 159
-
Agrees
Local forest microbes instead of bought ones Cho collects IMO from local leaf mold and warns that purchased microbes cost farmers money. Chun closes on the same contrast: IMO comes from natural forest compost, commercial biocontrol agents cost money.
Cho, IMO and Vital Power of Crops and Livestock, p. 21
-
Goes further
Counts drop at IMO4 Cho makes IMO4 by mixing IMO3 with the field's own soil so the microbes 'assimilate' with the soil's native ones, not to raise numbers. Chun's tenfold drop at IMO4 is the first count we have of that step; what it means for assimilation is untested.
2018/Cho - advanced hawaii/cho advanced 2018 2-3 @ 1:00:58 (translated)
-
Unclear
Which fungi are in IMO1 Cho's IMO1 is leaf mold with white hyphae. Chun found Aspergillus dominant in his IMO1 and more Rhizopus by IMO4; Cho does not name species, and Chun's IMO1 was refrigerated before use.
05-enzymes-and-microorganisms — Leaf mold from the forest
“I do not know natural farming very well, but I start to learn natural farming.”
▶ 0:08
“But natural farming thing is your own natural, and it comes from natural forest compost. So that maybe makes difference.”
▶ 24:25
In this talk
- 0:00A plant pathologist new to natural farming
- 1:38How biological control began
- 3:54Kinds of biocontrol agents
- 7:21How biocontrol works
- 8:06Free microbes from Korean extension centers
- 9:34Rice seedling disease in water-seeded rice
- 11:00Carbon sources that feed the bacteria
- 12:44Phytophthora root rot of pepper
- 14:07Tracking bacteria in rice roots
- 15:38Microbes in natural farming soil
- 19:33Microbe counts from IMO1 to IMO4
- 20:56Which fungi: Aspergillus to Rhizopus
- 23:24Trichoderma, and IMO versus compost
Transcript
Transcribed and checked by hand against the recording. Click any time to play from there.
0:00 I think I'm the last one, the one last speaker. I'm not really, I don't know, I do not know natural farming very well, but I start to learn natural farming. And I would like to talk about natural farming in the view of plant pathologies, plant diseases. So I made a title, Biological Control of Soil-borne Diseases and Natural Farming. Like the previous speaker, Kim Eun-ja, Eun-ja Kim told that she wanted to present next year after she completed many works.
0:53 It was the same to me. I had maybe two or four slides of IMO study to understand the underlying mechanism of the natural farming in terms of the disease control. But Professor Kim, [unclear] me pretty much. So I made time and I made a few more slides and added some slides. I may take maybe a little more time, maybe five in this case, not too much. So I would like to talk about this biological control and how it might be linked to natural farming.
1:40 And the biological control became popular in 1980s in America. Before that, people used a lot of pesticides to control pests and plant diseases, and it made a lot of environmental problems, problems. As you know, DDT is one of the examples. So Rachel Carson made a report, wrote a book, Silent Spring. So after congressmen, so after American people read that book, they need to know that we have to control
2:26 this pesticide. And then there was a famous scientist, James Cook. He was a USDA ARS research scientist, and they grow wheat in America, northern area of America. They grow a lot of wheat, and wheat has take-all disease, very severe. So the name of the disease is take-all. And then after they grow the wheat for five years, all of sudden this disease become less and less and they want to know why it is that so
3:13 and they found out that it was due to the Pseudomonas species, which is bacteria. So they called it disease-conducive soil, or disease-suppressive soil. Because there was that time that scientists tried to emphasize that we have to do sustainable or integrated pest management. Now it becomes, in Korea, environment- friendly agriculture. Now I think it is now developed into the natural farming itself. So the biological control is accomplished through many ways, and first
4:03 they started with one specific organism, like a Pseudomonas species, which controls take-all disease; Gaeumannomyces graminis is the fungus name. And then they started, so they started the bacteria which is very usually they talk about antifungal bacteria, so they could control plant disease. They look for this bacteria from [unclear], and then they look for maybe not just bacteria, maybe fungi. So they are looking for the Trichoderma species. This is very famous and developed as commercial commercial biopesticide, I think, worldwide. And another one, they looked at
4:51 Pythium oligandrum to control damping-off plant disease, and also non-pathogenic Fusarium. You know, Fusarium wilt is the famous disease, and this non-pathogenic Fusarium study was done in Japan specifically. So they used non-pathogenic Fusarium species to control Fusarium wilt, and also they used this one to control nematode, not just the fungus type, but nematode. So they called all these mycoparasites. And it was 1980s, or 1980s and 1990s. Then there was a famous scientist in Auburn University, Kloepper.
5:39 He suggested that there is plant growth-promoting rhizobacteria, which control plant disease, also promote plant health, plant growth. Now we know that this bacteria can induce systemic resistance, make plant more strong to plant disease, like similar to the immune system in animal. And nowadays, people are working on endophytic bacteria, which reside inside of the plant, inside of plant itself. Then they fix nitrogen, not like a nodulating rhizobium, but they fix nitrogen, and they also produce auxin, which is the plant hormone. So now, after
6:27 they started on this one around that time people started about organic amendment, which is compost, which might be very similar concept to natural farming. They pile up all the plant residue and ferment it and put it into the soil, which there is a pathogen, plant disease, and the disease seems to be controlled. And not seems to be statistically significant. Scientists always make a statistical analysis and then you see, is this significant or not so they made all kind of organic method the plant residue with the chicken litter and cow dung, and cow dung compost, all
7:11 sort of things. It's about biological control. And actually I do not have much slides, so I want to show what I have been through, just some of the slides for specific disease. This is the mechanism: the antibiosis; site competition for the infection site; and siderophore: the bacteria produce iron chelators, so the pathogen cannot use iron, so that pathogen's growth does not grow very well; and nutrient competition, and systemic induced resistance,
7:59 which I talked about already. And this one, out of this, this is a little out of this topic few years ago I suggested some proposal to the government. And in Korea there is a word for microbes: effective microorganisms. And I think this term was originated from Japan. Korean people try to use this effective microorganism. One of them is the yeast used a lot. So in this case, the Korean Agricultural Technology Center, many cities have all these fermentation facilities. They grow these bacteria.
8:45 and even for the cow, livestock, and they distribute to the farmer for free. So farmers can take this product without any money. They get this one and then spray to the crop field. So it's popular now. They use not only EM, which might be yeast, but there could be more than yeast. But the photosynthetic bacteria, maybe cyanobacteria, any kind of bacteria, all kinds of biological agents, some bacteria, or fungi.
9:34 And this is my study. When I came here, this is the first time I had to start something. In America, they produce rice somewhere, not everywhere, planting rice seed through the airplane. And Louisiana is one of those states. And in case of Korea, the Hyundai Asan company, they have a big farm. They plant rice seed through the airplane and grow the rice. In that case, seedling diseases occur often. And in case of Korea, for not a big farmer, if the farmer is too busy to do something, they plant rice seed just by hand.
10:20 And in certain cases, when the weather is very cloudy, no seed emerged at all. All these red color seeds are dead. And all those [unclear] seeds are dead. So I started to think that maybe there might be some bacteria that could control this kind of disease. And I just kept some pathogens as very severe pathogens. And then at that time, this idea is that if I provide some nutrient to the bacteria, biocontrol agent bacteria, maybe it controls plant disease better. And it shows that all these, this is bacterial biocontrol agent, and this is not inoculated,
11:09 and this one has inoculated one but this one was not severe but it was significant and this is how I had some concept that this is bacteria these are different carbon sources, and the bacteria used alanine very well, and [unclear] very well, but the fungus uses this kind of [carbon source] very well, but they do not use sorbitol or galactose or lactose. So I want to show that there is difference in terms of the carbon utilization between the fungi and the bacteria and we added this concept to seed coating of the rice seed.
12:03 And we had a group which is non-inoculated, so no Pythium. And here is a disease development. The base means that there is some formulation, Pythium, the fungus, but compared to this one, we add alanine or mannitol or sorbitol, which the fungus cannot use, they control it very well. And I will skip; it shows good results in seedling emergence, and also in the field, the plot which is [unclear] square meters, it shows the good yield. And in the pepper,
12:55 [this one] is control. The Phytophthora root rot is developed. They do not apply any bacteria or [unclear]. And this one, they applied bacteria and treated, and looks much healthier. And I added the same concept. The arabinose is used very well, but Phytophthora capsici, which is the pathogen of the root rot of the pepper, does not use it very well. So I added those ones. And this, Phytophthora capsici, has severe, say... no, I'm sorry, this is non-inoculated control, and no disease at all.
13:47 Phytophthora capsici, the plant pathogen, alone, 81%. Almost all the plants got disease, about 80 percent. If you add bacteria, it is controlled, but if you have those specific carbon sources, they control better. And this one, this slide is for understanding what happening what happened after we inoculated bacteria how the bacteria moves and we made some bacteria which can fluoresce in green color So this is a wild-type bacteria, which induces systemic resistance. Without
14:37 transformation with the green fluorescent gene, you cannot see any green color under fluorescent microscope. But this is the mutant that has green fluorescent gene. And if you grow rice seed in hydroponic culture, this bacteria goes inside. This is the dynamic inside, and they only lose a little bit. And [in] seven days, [unclear]. Okay, I finished. All those works are to develop
15:26 the better biological control agent, to understand some mechanism, how the bacteria attach the rice seed, root, very well, how it goes, how it stays long. And now, just this year, I started to study on indigenous microorganisms in natural farming soil, which Professor Kim's students studied in the Paju farm. And I brought those soil, actually they brought that soil, and I [studied the] population of those one. And I would expect that maybe there are more diverse bacteria
16:20 or more diverse fungal population in IMOs or natural farming soil. But I cannot make a conclusion yet, but it was not really what I thought. Now, this is the word which you use often: IMO. So I learned... I asked this one often to Professor Kim: what is IMO, and what is the indigenous microorganism. And when I talked with Chris Trump, maybe it is natural forest compost under the tree, because there
17:05 is a natural population of the fungus and some bacteria too. So the IMO 1 and IMO 2, and IMO 3 and IMO 4. And this soil must be originated from the [unclear] where they want to grow the crops. And I started it. So this is per gram of soil, the colony forming unit, which is, say, the number of the body of the bacteria or the fungi, times 10 to the 5. And this is control soil. I think this is, they grow the peanut, Zacheous's experiment.
17:51 And so control soil, natural farming soil, conventional soil, organic soil, there are a lot of bacteria in control soil, but less bacteria in natural, or conventional, organic. We do not know exactly how it happened, and we are doing replicate again. We did replicate, but my students and Professor Kim's students had different results. Why it is so, I have to check again. But it's reliable. Anyway, there are more bacteria, more fungi, and we cannot determine whether to do it. Anyway, number doesn't mean [unclear].
18:38 So maybe natural, I thought natural farming soil has more, more of the fungi. That's what I thought, because you add rice bran and those ones. But maybe not. And this is natural, again. We are doing the second experiment. And we just focused on the fungi. Natural soil, 18; organic, 13; and conventional, 7. So there is more fungi in natural soil in the second experiment. But this number, 18 times 10 to the 2, 18 times 10 to the 2, in terms of micro population,
19:29 may not be significantly statistically different, but we have to check again. But this slide says something about IMO things. This we started IMO 1, IMO 2, IMO 3, and IMO 4. This is maybe not really fresh because IMO 1 we brought it and put in the refrigerator. Maybe two weeks later we started it. But IMO 4 maybe made it more fresh. But anyway, say that IMO1 has a lot of bacteria, 10 million times 151, so 10 million... maybe 15 billion bacteria. And fungi, 10 million times 56, a lot of fungi.
20:18 IMO 2 is decreasing. And IMO 3... IMO 3 is decreasing. IMO 4, because we mix 1 to 1 ratio with soil. So compared to this one, it's statistically much... 100 times less... Not 100 times, 10 times less. this is about 10 times less too. So the population is decreasing pretty much. So then I could not study yet the diversity of the fungal population between two
21:11 natural farming soil and organic, control soil, and fungal population diversity among the IMOs. Then there was some difference when compared to IMO 1 to IMO 4 when it goes this much. But in this case, for fungi, we do not know about the bacteria, we cannot identify bacteria yet. So fungi, there are a lot of [unclear], sporulating fungi, Aspergillus and maybe Trichoderma, but Aspergillus was dominant. This kind of color, there is Aspergillus, which has spores.
21:59 And then, when it goes to IMO4, which is this population, then there are more Rhizopus fungi. Rhizopus fungi is zygomycete, which is called also dung fungi. And most of the, almost most of the Rhizopus fungi does not cause plant disease at all. But it grows so fast. It is similar to maybe one of the black, black [unclear], usually the decayed [unclear] by this [unclear]. They grow much faster than this one and they have black spores. It produces spores too, but maybe this one has more spores.
22:44 It does not, the height... it does not go faster than this one. This one is far faster than any other fungus. [unclear] grows very slow. They grow slowly. So maybe the component of the fungal population might be changing from IMO 1 to IMO 4. And maybe natural farm soil may have different component of the fungal population compared to traditional compost. So we can recommend it. but we have to start I cannot make any conclusion not yet but we have to study more and more. And this is Trichoderma. This one is also slower than the
23:31 Rhizopus. Anyway, this is a famous one, and many products now available for those [uses]. So I say, what is difference between the compost and organic amendment, or compost and natural farming thing. The source is different. The source comes from natural forest. So there are maybe mostly no pathogens during this treatment. But the organic amendment, they usually [use] crop residue, which is leftover and mixed with all kinds of, if you add the chicken litter and the cow dung, it will be all kinds of bacteria. So there might be difference. And otherwise, later on, organic amendment,
24:13 the biological control agent, maybe... Maybe if you use continuously, better. But those one cost money. But natural farming thing is your own natural, and it comes from natural forest compost. So that maybe makes difference. But we have to figure out that one later on. Hopefully in the future. Okay, this is it.