A recent report in Science (Vol.359, 1399-1403) describes how a single fungal gene controls plant cell-to-cell invasion by the rice blast fungus. It is an interesting description of how a plant pathogen manages to invade a plant cell and manages to travel cell-to-cell, evading the plant immunity system, eventually killing significant sufficient tissue to sporulate and spread to other plant tissue. This study involved a lot of biochemistry and microscopy and uncovered interactions that are probably common to other fungal-corn interactions.
Living plant cell walls have microscopic holes, called plasmodesmata, that allow passage of sugars and proteins to adjacent cells. These holes are smaller than normal fungal filaments (hyphae). In this study in which the pathogen Magnaporthe orzae, cause of rice blast disease, initially invades the plant by forcing the outer leaf cells with an appresorium, to occupy a cell but maintaining the cytoplasm in the cell. The hyphae of this fungus reduce the size of the hyphae to about 1/10th to squeeze through the plasmadesmata into the next cell. The plant cell resistance includes reacting to the presence of the invader by depositing callose to close the plasmodesmata, and therefore restricting the fungus to the initially invaded cell. The researchers found that a single fungal gene delays the resistance reaction until the pathogen has passed on the next cell. Mutants of this gene in the fungus are not able to pass onto the next cell. Resistance is related to a quicker reaction in closing the plasmodesmata as well as repression of the fungus within the infected cells.
This study involving the interaction between a fungal pathogen and host is probably common to many leaf diseases. Relatives of this fungal species attack other grass crops such as wheat and barley but apparently not corn. The study illustrates the evolution of methods of attack by pathogens and competing defense systems by plants. It is also interesting that the study was done by several specialists employing multiple techniques and understandings of their science.
When corn seed, with its own biological potential problems, is placed in soil populated by multiple micro-organisms ready to feed on organic matter, multiple battles begin. As the seed imbibes water, internal membranes swell, causing some leakage of stored sugars and proteins into the soil. Many soil organisms grow towards the damaged seedlings, attracted by the leaked substances. First tissue exposed to the potential invaders are the primary root and mesocotyl, two tissues that are later disposed when the secondary roots take over for the main root functions. Invaders are detected chemically by the corn cells near the invasion, turning on local production of resistance antibiotics. Success in suppression of the invaders is affected by aggression and intensity of the potential pathogens, environmental effect on pathogens and seedling and the biological vigor of the seedling.
Several fungal species are associated with seedling root rots. Fusarium, Penicillium, Pythium and Rhizoctonia species are among the most prominent fungi found in diseased corn seedlings.
Fusarium species are common in nearly all soils with any organic matter. Every corn seedling is exposed to this fungus and yet only a few show symptoms. The actual cause of the seedling disease is usually more complex than simply identifying the fungus present in the corn tissue. Analysis of the cause should include consideration of environmental factors such as water concentration and temperature, and seed germination quality. Vigorous seedling growth can quickly dispose of primary root and mesocotyl dependency as secondary roots develop and new leaf tissue produces the quantity of resistance factors needed to control potential invaders such as these fungi.
Seed treatments can give the seedling some temporary relief from fungal invasion pressures. Genetic variability within the pathogen population can weaken its affect in some circumstances. Vigorous early growth due to corn hybrid genetics and seed quality favor escape from seedling disease pathogens even when environments are not optimum for early corn growth.
Corn seedlings are frequently attacked by a fungal-like organism of the Pythium genus when the soil is extremely cool and wet. Pythium belongs in a group of organisms called Oomycetes. These were once considered fungi, but more recent research has shown that they are more related to algae. Other pathogenic oomycetes include those causing diseases such as downy mildew of several grasses (including crazy top of corn) and Phytopthora root rot of many crops. Oomycetes were considered fungi because they usually produce filaments and spores plus they absorb nutrients from plants and animals. They differ from fungi by mostly lacking individual cells within the filaments, resulting in many nuclei within the long filaments. Most fungal cell walls are composed of chitin whereas the oomycete filament walls are cellulose, a difference that relates to the host resistance system. Often the first signal of fungal invasion is of the chitin, triggering the production of defense systems. Detection of an oomycete invasion requires a difference in plant chemistry.
Oomycetes frequently produce swimming spores that are released from overwintering spores (oospores) in the soil. Pythium spores, with flagella, swim to host root cells on which they grow hyphae to invade the host. Consequently, they cause the biggest problems in fields that are temporarily flooded with heavy rains and cool conditions. Studies have shown that 55°F is optimum for most Pythium species infecting corn in Midwest USA. The low temps probably slow down the host plant’s growth rate and inhibit potential competition from soil fungi.
As many as 18 species of Pythium have been shown to be pathogenic on corn. Often, they destroy the outer layer of the new root tissue. If this occurs before secondary roots become the main source of water uptake, the seedling will wilt. Although seed treatments can offer some protection, genetic diversity within a species often includes resistance to most treatment chemicals. Pythium genetic diversity also includes ability to attack rotation crops such as soybeans. Pythium species are easily isolated from soil but completely accounting for all the species or diverse genetics in regard to effective seed treatments or genetic resistance is not easy.
Control of the disease is best done with controlling water holding capacity of the soil.
Germination testing, especially the cold test, is probably the best predictor of successful emergence in the field. Comparing lab results with field emergence percentages is not easy because of the other variables involved in success. Cold tests are the best to reflect growth in the low temperatures that most corn seed faces when planted in temperate zones. Damage or weakness of cellular membranes during imbibition will show that some seed are not germinating as quickly as the less damaged seed.
Slight differences in soil micro-environments within a few feet in the field can have a drastic effect on the weaker seed. Tighter soils can have oxygen deficiencies, higher water content and more resistance to growth of the coleoptile as it pushes upwards. Minimum tillage debris on soil surface prolongs the colder temperature. Small differences in planting depths and consistency of seed drops with planters also make field assessment of field germinations of multiple seed lots difficult.
Ideally a germination lab should consistently compare its results with field emergence. This is not easy because such a test should include multiple replications within a field and across several fields. Comparison of lab tests on referee samples by 20-40 public and private labs usually show warm test variance of 4-5% on generally high germinating seed and of up to 10% on cold tests. Some of these are ‘official’ labs that prescribe to similar methods but subtle differences between labs, or even among samples apparently account for the differences in results. The complexity of comparing with field results further inhibits the development of the perfect lab test for predicting field emergence in all conditions. On the other hand, poor performance in most cold tests suggests that a seed lot is more likely to have field emergence problems. A company eventually attempts to establish acceptable cold test result standards from a lab that is most frequently associated with reliable field emergence. It is important that the standard is relevant to that lab tests and not necessarily to other labs.
Seed companies and growers agree with the desire that seed germination quality should not be a factor in performance of the hybrid but predicting this is not perfect.
Corn seed quickly imbibe water when exposed to its maximum within 3 hours, regardless of the temperature. Imbibition allows the swelling of membrane-bound cellular contents such as mitochondria, plastids and ribosomes. Metabolic activity in these organelles is dependent upon temperature and status of those membranes after imbibition. Some individual seed within a seed lot are more vulnerable to membrane damage, especially from the sudden swelling due to imbibition. Membranes do have the ability to self-repair as new metabolic products are activated. This repair is more rapid at higher temperatures (70°+F) but can be inhibited or very slow when temperature is near 50°F.
The standard warm test in the USA was originally designed to measure the percentage of seed that are viable and thus is conducted at above 70°F for 7-10 days. The problem arises when some individual seed being tested manage to show partial germination, with only the shoot or root showing. Others may have shoots and roots eventually growing but much later than the others in the sample. There are ‘official’ definitions of approved germinating seed but labs still need to have some way of defining these slow or injured plants. Often, they become defined as ‘abnormals’.
Even with attempts to regulate and define germinations in the warm test, and with defined methods such as substrates used and amount of water added, there remain differences among labs. Much of that is related to definition of these delayed germinating seedlings.
Cold tests are designed to predict the reality of emergence in the lower temperatures that are common to corn planting dates in the temperate zones. Usually the seed is placed immediately after watering in a 50°F environment for 7 days. The intent is to allow expression of damage that is likely to occur in the field at the earlier planting dates. Under these conditions most viable seed with little cold imbibition damage will germinate well when placed after the cold 7 days into a warm 70°F+ environment. Those seed with damage, however, will either not germinate or will be significantly delayed. Labs differ in substrates used for the cold test. Each has some difference in results and consequently companies may establish different standards for release of seed lots to their clients.
The ultimate objective of seed germination testing is to predict if seed germination quality will detract from maximum genetic expression for performance of the hybrid in the field. Very few seed lots will show 100% germination in cold tests. There are other factors influencing final plant stand and uniformity of growth in the field, but germination tests are intended to limit this factor.
The challenge is to identify the seed lots that will have sufficient germination in the field 4-6 months after lab testing. Real life biology is not always easy.
Corn seed ages, like all of us, but not every seed within a seed lot nor every seed lot of a hybrid nor every hybrid age at the same rate. These principles are well known but the seed industry still has battles to manage this natural process. Most of the ageing process is believed to be occurring at the membrane level where the metabolism activity is occurring even at a low level when seed is dried below 13.5% moisture. Membranes of major cell organelles responsible for respiration also need some upkeep to retain function. Environment in the field and later in the seed processing plant can affect the integrity of the membranes in the seed prior to drying. It is established that most modern genotypes benefit from rapid drying from air movement rather than high temperatures, but outside environments can influence meeting that goal.
Most seed are dried to 11-12% moisture, providing some leeway for individual seed differences within a seed lot. Long term storage of seedstock parent seed is sometimes dried to 7-8%, without notable drops in germination for a few years. Adding slight amounts of moisture, not enough for germination but enough to accelerate cellular respiration, can lead to a drop in germination percentage. I have not found research studies on this subject, but 3 personal examples come to mind. I witnessed a company retreating carryover seed with intent of maintaining the germination quality, but the result was a drop of germination. Perhaps it was from handling damage or perhaps from moisture addition. Another case that I had witnessed was when a company in a humid environment added a seed treatment that resulted adding some moisture to the seed but did not dry the seed immediately, resulting in a lower germination.
Several years ago, a seed company wanted to test our ability to distinguish ‘selfed’ seed from hybrid seed, by adding colored seed treatment to cover up the different seed colors originally on the female parent seed from the hybrid seed. We reported the selfs but also a low germination. After discussion, the customer sent us the original seed without the extra seed treatment, and we found a higher germination. Some seed treatment components can be damaging to seed but adding a small amount of moisture from the seed treatment solution has potential to reduce germination, perhaps pushing a few vulnerable individual seeds over the edge.
Given all the potential problems in producing and maintaining high germination percentages, it is amazing that our seed industry provides high quality seed to their customers.
Corn seed, dead or alive, will allow water to enter through the pericarp, causing the kernel to swell. Dry cells in the embryo retain many membrane-bound structures including mitochondria, plastids including chloroplasts, nucleus and endoplasmic reticulum. Cellular membranes are composed of phospholipids and proteins organized in a manner that regulates the biological function of the cell organelles including regulation of movement of products in and out of the organelle.
Membranes in a dry corn seed cell are only slightly active, oxygen to pass through, for example but have more of a gel like structure. Within a few hours of imbibition, the structure changes as the phospholipids become moist and swollen. Resulting metabolism with activation of respiration in mitochondria, fueling gene translation in the nucleus, movement of RNA on the endoplasmic reticulum and production of protein in the ribosomes. The water plus metabolism causes the radical part of the embryo to elongate and the germination process has begun.
Two potential problems can stop this process. The seed may no longer have sufficient structural integrity, possibly because the aging process while dry no longer maintained the metabolism needed for maintenance. A second problem can be that the imbibitional process caused breaks in the membranes that were not adequately repaired during those first few hours of swelling as water moved into the cells. Membranes do have the capacity to self-repair and often do when metabolism is active. However, this process is temperature related, and in corn this repair process is very slow when temperatures are at about 50°F. Imbibitional chilling injury is the term used to reflect poor germination of some seed when planted in cold soils.
Every seed within a lot, although genetically identical, has had a slightly different environment experience. Location on the ear, exposure to insect or fungus, location in the drier, handling in the sheller or bagging processor all could affect its tendency to cellular injury. This usually if most profoundly expressed when it imbibes water under cold conditions. Some seed may reflect this by only delaying the germination as it repairs sufficient membrane for metabolism to germinate although later than the other seeds.
The corn kernel is a fruit with one giant seed. We humans mostly bred and selected this grain for its use as a food source, increasing the endosperm size with carbohydrates. Selecting for desirable seed traits has been at least somewhat secondary to the grain production. On the other hand, uniform and reliable field emergence is a major contributor to corn grain production in modern corn hybrids. This is dependent on the science and experience of seed producers.
Much of the propensity for high germination is dependent upon the female seed parent. Pericarp, being totally part of the female plant, affects rate of moisture loss during drying, vulnerability to physical damage from handling of the seed and susceptibility to ear molds. Mitochondria genetics are totally inherited from the female parent. Much of the damage from rapid, cold imbibition of water at the initial stage of germination involves the mitochondrial membranes.
Maize kernels handled as grain need to be stored at 15% moisture to avoid mold. Modern hybrids are usually allowed to dry in the field well beyond the 30-32% moisture level that black layer forms and completion of movement of carbohydrates to the kernel. It is usually most economic to allow drying in the field before finishing with artificially drying.
Most maize seed begin losing germination capacity if left in the field during those final days of slow drying in the field. Studies have shown that greater germination percentages are retained if seed is harvested in the 35-40% moisture level and then is quickly dried with lots of air and less than 100°F. Retaining the higher moisture level for some time initiates metabolism in the embryo, essentially an artificial aging process. Seed dried to about 12% moisture is considered optimum of storage and retention of high germination rates.
Producing and retaining high germination is the result of research of each hybrid parent’s vulnerability as well as experience with weather and facility. Drought damage during grain fill, rain delaying harvest, drying at too high temperature and not enough air, rough handling during processing and adding too much water during seed treatment can contribute to below standard germination. One can write a manual for production of seed corn but ultimately it takes some experience to apply the science. Like most agriculture.
Another Fusarium species, Fusarium graminearum, forms a sexual stage of the fungus when its mating types combine. That sexual stage is identified by the name of Gibberella zeae. This fungus is associated with Gibberella stalk rot and wheat scab. The fungus is common in corn debris, producing huge numbers of Fusarium spores during much of the corn-growing season. Spores germinate on the silks, with the fungal filaments growing down the silk channel towards the ovule. Generally, if pollen tube growth reaches the ovule first, the following collapsing, dry silk tissue effectively stops the fungus. Silks are most vulnerable during cool, wet weather as pollen spread is poor but fungal spore production is high. This results with a prolonged period of silk exposure. Later infection of the kernels appears to be related to kernels physically injured by hail or insects. Husk leaves tightly wrapped around the ear also appears to be related to spread of this mold within the ear.
Early infected kernels will fail to develop completely, will be light in weight and often will not germinate if planted as seed. Later infection can spread to cover much of the ear with a mold as the fungus spreads from the initial infection area. This mold produces mycotoxins including deoxynivalenol (DON). This toxin is associated with severe health problems in swine. The fungal spores produced among the grain also can be detected by swine and cows, causing them to reject the grain. I recall a personal situation in which I was a sent a corn sample from seed dealer who claimed that newly harvested grain was rejected by pigs and calves. Observation of the grain in my lab showed lots of Fusarium spores- so much that I asked to see the storage bin where the grain was stored. A single hand full of the grain from the bin revealed a cloud of spores. The grain was dried in the bin by air being blown from the bottom up through the bin. This air, however was moved over an accumulation of previous year’s debris below the grain. A sampling of that debris revealed that it was heavily infected with Fusarium. The new crop was being inoculated with the fungus as it was being dried.
DON has been shown to accumulate in grain stored at moisture higher than 20.5%. One should assume that the fungus is ubiquitous and that monitoring the grain drying and storage is important to avoiding this toxin problem.
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About Corn Journal
The purpose of this blog is to share perspectives of the biology of corn, its seed and diseases in a mix of technical and not so technical terms with all who are interested in this major crop. With more technical references to any of the topics easily available on the web with a search of key words, the blog will rarely cite references but will attempt to be accurate. Comments are welcome but will be screened before publishing. Comments and questions directed to the author by emails are encouraged.