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The distinction between colonization and sporulation in evaluating species composition, viability and reproductive potential was discussed in the last newsletter. Several users have raised questions about situations where species composition appears to change over time or with a change in culture conditions. In most of these instances, we think the relationship between colonization and sporulation greatly determines what we see and how we interpret those observations.
The occurrence of sporulation in a culture provides positive evidence of colonization by a glomalean fungal organism. Conversely, however, the absence of sporulation does not indicate absence of colonization. At soil P levels where mycorrhizal and nonmycorrhizal plants exhibit equivalent growth responses, for example, we have observed that sporulation ceases completely but colonization still is maintained albeit at a much reduced rate. We have a strong suspicion that a threshold level of colonization must be reached for spore production to be induced. Logically, interdependence of colonization and sporulation would seem to be a function of carbon partitioning in source-sink relationships. These factors are likely to differ among isolates (populations). Two major determinants of these kinds of interactions are:
(1) impact of host-soil-ambient environments on the growth of an individual organism, regardless of how many different species might be present
(2) competition among two or more organisms co-inhabiting root biomass. The former determines success of monospecific cultures; both determine composition of trap cultures containing mixtures of species.
I. Environmental Factors
Once an organism of a species has been isolated and propagated in pot culture, success in maintaining viability and fecundity of that fungus depends on the culture environment as well as inherent genetic variables. We have observed that some fungal isolates are abundant sporulators (and colonizers) when each is alone in the first few subcultures. Then, spore numbers decline (with or without a decline in colonization). For example, A. delicata AZ101 is an isolate obtained from Mike Pfeiffer, who already had established the fungus alone in culture. This isolate was highly fecund at WVU in three successive propagation cycles. Then, in the fourth cycle, spore numbers precipitously declined to half former levels. By the sixth cycle, we were obtaining fewer than 40 spores/50 cm3 of culture medium. Part of this behavior may be due to a response of the fungus to prolonged homogeneity of host, soil, moisture, and temperature conditions relative to “typical” variation found in the field.
Viability and fecundity also can increase when the culture environment favors the organism. For example, A. koskei WV768 produced fewer than 40 spores/50 cm3 of culture medium when first cultured alone. After three propagation cycles, the isolate was producing 400-600 spores/50 cm3.
II. Competitive Effects
Competition for carbon and inorganic nutrients likely is quite dynamic for different parts of a glomalean fungus, depending on rate of carbon allocation and substrate availability. Some workers have the mistaken notion that glomalean fungi (like many other fungal organisms) sporulate in response to stress or some imposed constraint on growth. Dynamics of sporulation in the field versus in pot culture suggest a different dynamic. Glomalean fungi, especially those in Glomus, Acaulospora, and Entrophospora, will begin to sporulate as soon as some colonization develops whereas others invest mostly in auxiliary cell formation early in mycorrhizal development (Gigaspora, Scutellospora). Sporulation then occurs at varying rates depending on fungus, host, and environment. One of the most important variables appears to be rate and duration of root growth. For example, in 20 liter pots containing one corn plant, sporulation may not be evident for several months whereas the same fungus and plant in a 150 cm3 cone-tainer will begin to sporulate profusely in less than 30 days. The common connection: sporulation increases rapidly once root growth has terminated as a result of pot constraints. The same occurs in the field. Sporulation of many species is low or absent, but roots are not bound within pots and will grow as a function of host phenological behavior and will be spread out over a full growing season. We think that this dynamic is a function of carbon allocation patterns to the part of the fungus with the greatest demand at any given time. When roots are actively growing, so is fungal hyphae and associated vegetative structures; spores are a luxury and will form only when there is excess carbon available (which may be partly why C4 grasses are better culture hosts than C3 grasses). When roots cease growth, so does fungal vegetative growth; shoots are still producing photosynthetic carbon and it now can be repartitioned to sporulation without any negative impact on the host (and especially in pots where fruit formation (another major sink in plants) is reduced or completely inhibited.
What happens when there is a mixture of fungal species trying to occupy the same root space? They all are likely to find room to become established (unless inoculum is layered and distribution of all available propagules is severely constrained) and then obtain enough carbon to survive and fluorish to varying degrees. For those fungi which establish most quickly and then grow aggressively, "possession is nine-tenths of the law" and they will be the first with sufficient excess carbon to sporulate. Even after plants have ceased root growth, only the dominant colonizers are likely to sequester the carbon to sporulate profusely. Poor colonizers may inhabit roots for numerous propagation cycles before sporulation is detected, and this would be a function of their growth relative to other fungal occupants.
Consider the following examples:
Gigaspora margarita FL105: In the first propagation cycle of this accession (FL105-1), G. etunicatum comprised 22% of total spores in the extracted population. To eliminate the G. etunicatum component, the next subculture (FL105-2) was produced from spore inoculum. Only Gi. margarita spores were produced in that and the next subculture (FL105-3). However, G. etunicatum reappeared in FL105-4. This pattern occurs often when we are not extremely vigilant in removing all hyphae, debris, etc. from spore preparations used as inoculum. We think that it only requires one hyphal fragment to establish one infection unit to keep the G. etunicatum isolate alive, and then this fungus increases inoculum potential over several cycles until it finally produces enough biomass and carbon sequestration to actively sporulate.
Scutellospora persica MD103: This accession contained a mixture of Gi. gigantea, S. verrucosa, S. weresubiae and G. intraradices spores. A second propagation cycle using whole inoculum (MD103-1) yielded the same fungi, but in different proportions (G. intraradices became the dominant sporulator). In the next cycle (MD103-2), spores of S. persica were detected for the first time. Since no other accessions in the collection contained this fungus, we concluded that S. persica was present in the parent culture but did not have the capacity to sporulate until conditions changed (greater inoculum potential relative to other fungi present, more biomass to increase carbon sink size, etc.)
Glomus species FL312: This accession originally contained only an undescribed Glomus that was a yellow-brown variant of G. etunicatum (we maintain some skepticism that color alone discriminates this as a unique species). In the first propagation cycle at WVU (FL312-1), only spores of A. morrowiae and S. heterogama were produced (no Glomus anywhere to be seen). Spores of these fungi were separated and used to establish monospecific cultures (FL312A and FL312B, respectively, to maintain a link to the parent culture). Rexamination of the original culture material showed no evidence of these two species. When FL312A was propagated for two more cycles using whole inoculum (pot contents), spores of the Glomus yellow-variant reappeared in about 30% of the total extracted population. When these spores were used to start a separate culture (FL312C), the fungus grew and was maintained thereafter as a monospecific accession. The material of Glomus FL312, when grown repeatedly in Florida, never showed the presence of these other fungal species, suggesting that their presence is masked by host-environment conditions that constraint spore formation. These fungi are likely present at low levels, but they never sporulate. When conditions changed radically to a different host and much cooler greenhouse conditions, the Glomus species failed to grow well enough to sporulate and the other occupant species quickly dominated. When setting up monospecific cultures, hyphae of the Glomus species was unknowingly carried along and this fungus eventually adapted sufficiently to the new conditions to regain enough biomass to sporulate.
Scutellospora calospora CL288: A low number of G. occultum spores were observed in the deposited material of this accession. They remained in low numbers in the next propagation cycle (CL288-1), but exploded in CL288-2. At the same time, S. calospora ceased to sporulate and could not be recovered in a subsequent culture cycle. In this case, one fungus (G. occultum) completely displaced another (S. calospora).
Acaulospora scrobiculata WI994: In the first culture cycle of this accession (WI994-1), a few spores of G. etunicatum were discovered. In WI994-2, 90% of the culture consisted of G. etunicatum spores and sporulation by A. scrobiculata was severely reduced. When spores of A. scrobiculata were separated and inoculated onto sudangrass seedlings, the fungus again sporulated profusely.
Glomus etunicatum UT316: The first generation culture (UT316-1) showed all indications of being monospecific. In UT316-2, a few spores of G. occultum were detected. In UT316-3, G. occultum comprised over 75% of the spores extracted. When G. etunicatum was cultured separately from spores, colonization and sporulation again were high.
Glomus etunicatum ND269:
The culture appeared monospecific after the first propagation cycle. Glomus
clarum was detected in ND269-2, but spores were few and they were so visibly
parasitized to be presumed dead. But we failed to take hyphal fragments into
account, and in ND269-3, G. clarum comprised 90% of spores in the culture.
When ND269-2 was recultured on two different occasions (and different times
of the year), the same result was obtained. These results suggest that despite
the presence of debilitated spores, highly infective propagules still were present
and the fungus was so aggressive as to quickly dominate in only one culture
cycle.
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