Habitat Life Style And Life Cycle

Neurospora (Figure 5.1) is frequently seen growing on stubbles of sugar cane after the canes have been harvested for milling and the agricultural field is burnt (Pandit and Maheshwari, 1996) to clear the trash of the cut leaves. In addition to the pink-orange type, a yellow-colored N. intermedia is found in Asia on maize cobs which have been discarded on road-sides, parks or railway tracks after the roasted kernels are eaten by residents. Another species, N. discreta—a species thought to be infrequent, limited mainly to Ivory Coast and Papua New Guinea—was found growing beneath the bark of trees damaged by wildfires in western North America, including Alaska (Jacobson et al., 2003). In the laboratory, the vegetative growth of all species of Neurospora occurs satisfactorily on a simple nutrient medium between 20 and 40°C. The reason for substrate preferences or differences in geographic distribution of species in nature is a mystery but could be due to their preferences for certain types of substrates for sexual reproduction. For example, N. crassa and N. intermedia show good fertility in media containing sucrose, whereas the N. discreta crosses are infertile on sucrose medium but satisfactorily reproduce on media containing filter paper cellulose. The yellow strains of N. intermedia, unlike the orange strains, reproduce poorly in media containing sucrose though they are very fertile on corncobs. A general observation is that conditions that favor sexual reproduction are different from those that favor asexual reproduction.

Thetmaphillic Fungi

Figure 5.1 Neurospora growing in nature. (A) Mass of orange conidia erupting through cracked epidermis of a burnt sugar cane stump in an agricultural field. (B) Conidia erupting at a node through the openings created by burning of the adventitious roots. A pencil was placed as a size marker. (See color insert following page 140.)

Figure 5.1 Neurospora growing in nature. (A) Mass of orange conidia erupting through cracked epidermis of a burnt sugar cane stump in an agricultural field. (B) Conidia erupting at a node through the openings created by burning of the adventitious roots. A pencil was placed as a size marker. (See color insert following page 140.)

The linear growth rate of Neurospora—up to 5 mm per hour—is one of the fastest among the fungi. The growth rate is easily measured in a "race tube" in which mycelium growth on an agar surface is confined to one dimension, allowing the position of the advancing mycelium to be marked at regular intervals (see Figure 11.6). Morphologically similar strains can be physiologically different. For example, the heterothallic species N. crassa and N. sitophila are comprised of strains of the opposite mating types, referred to as A and a, which can be crossed (mated) to form structures (fruiting bodies) called perithecia that enclose asci bearing meiotically formed ascospores. In general, the species produce eight ascospores of which four each are of the two mating types. Barbara McClintock, who discovered mobile genetic elements (transposable elements) in maize and a discovery for which she received the Nobel Prize, also studied Neurospora. In 1945, using acetoorcein staining and light microscopy, McClintock showed that Neurospora has seven chromosomes (haploid number n = 7, meaning it has one chromosome of each type and therefore one copy of every gene). The chromosomes are numbered based on their length and this numbering based on their size does not correspond to the numbering of the linkage groups. The term linkage group refers to an order of linked genes whose linkage has been determined on the basis of recombination frequency. The numbering of linkage group is in the chronological order of their discovery. Linkage groups correspond to chromosome numbers but may not correspond to the numbering of chromosomes based on the basis of their size. For example: LGI = chromosome 1, LGII = chromosome 6,

LGIII = chromosome 3, LGIV = chromosome 4, LGV = chromosome 2, LGVI = chromosome 6 and LGVII = chromosome 7. The chromosomes can be separated by pulsed field gel electrophoresis (PFGE). The total DNA of N. crassa is 43 megabases; the largest and the smallest chromosomes are 10.3 and 4 megabases, respectively.

The life cycle of Neurospora crassa based on laboratory-grown cultures is shown in Figure 5.2. The sexually (meiotically) formed ascospores of Neurospora are constitu-tively dormant propagules that can survive in soil for several years. The activated ascospores germinate and form mycelium which produces two types of asexual (mitotically

Neurospora Life Cycle

Figure 5.2 Life-cycle of Neurospora crassa. The ascospore (1) germinates after chemical or heat activation and forms mycelium. Asexual reproduction occurs by formation of mitotically-formed inconspicuous, uninucleate microconidia (2) or 2-3 nucleate orange-colored macroconidia (3) which are dispersed. Sexual cycle begins with coiling of hyphae around ascogonial cells from which a trichogyne projects out formation of protoperithecium (4). The trichogyne picks a nucleus of opposite mating type from microconidia, microconidia or hyphal (stages 5 and 6). Nuclear fusion (stage 7) leads to formation of asci containing 8 haploid ascospores of the two mating types, A and a. From Perkins et al. (2000). With permission from Elsevier.

Figure 5.2 Life-cycle of Neurospora crassa. The ascospore (1) germinates after chemical or heat activation and forms mycelium. Asexual reproduction occurs by formation of mitotically-formed inconspicuous, uninucleate microconidia (2) or 2-3 nucleate orange-colored macroconidia (3) which are dispersed. Sexual cycle begins with coiling of hyphae around ascogonial cells from which a trichogyne projects out formation of protoperithecium (4). The trichogyne picks a nucleus of opposite mating type from microconidia, microconidia or hyphal (stages 5 and 6). Nuclear fusion (stage 7) leads to formation of asci containing 8 haploid ascospores of the two mating types, A and a. From Perkins et al. (2000). With permission from Elsevier.

derived) spores. Under conditions of high-sugar and nitrogen availability, the fungus produces powdery pink-orange (macro) conidia that are 5 to 9 ^m in diameter and contain 2 to 6 nuclei, whereas under low-nutrient conditions the fungus mainly produces inconspicuous, uninucleate microconidia that are 2.5 to 3.5 ^m. The microconidia function as male cells and are formed simultaneously with structures called protoperithecia that are formed by the coiling of hyphae around ascogonial cells, one cell of which acts as the female gamete. DNA sequence analysis shows that the two alleles of the mating type locus, mat A and mat a, or A and a, have highly dissimilar DNA sequences and for this reason they are termed idiomorphs (Metzenberg and Glass, 1990). Mating occurs only between strains of A and a mating types—that is, between individuals that are sexually compatible. A slender hypha called the trichogyne projects out from the protoperithecium and is attracted toward macro- or microconidium of the opposite mating type, indicating that recognition of a mating partner is based on pheromone and pheromone receptor. The nucleus, picked up by this trichogyne (Figure 5.3), migrates to the ascogonium where fertilization occurs. In the laboratory, crosses are made by adding macroconidia (because these are obtained easily) from one culture to a culture of the opposite mating type that has been pre-grown in a medium with low nitrogen and carbon to induce the formation of protoperithecia. The haploid nuclei of the opposite mating type fuse in a hook-shaped structure called a crozier. The diploid zygote nucleus does not undergo divisions; rather, it immediately undergoes meiosis. The not-readily noticeable ascospores are formed at a different time from the striking conidial phase and were therefore missed for a long time.

Figure 5.3 Chemotactic growth of trichogyne of Neurospora crassa. (A) Microconidia (male cells) were placed at lower left side. (B-D) Curvature of trichogyne is a visible evidence of production of a pheromone by conidia. Reprinted with permission from G.N. Bistis (1981), Mycologia, Vol. 63, Number 5. © The Mycological Society of America.

Life Circle Fungi Neurospora Microconidia

Figure 5.3 Chemotactic growth of trichogyne of Neurospora crassa. (A) Microconidia (male cells) were placed at lower left side. (B-D) Curvature of trichogyne is a visible evidence of production of a pheromone by conidia. Reprinted with permission from G.N. Bistis (1981), Mycologia, Vol. 63, Number 5. © The Mycological Society of America.

In Neurospora, the four haploid nuclei, produced from a single meiotic division, divide mitotically to produce eight nuclei that are individually sequestered into eight oval-shaped cells called the ascospores and are enclosed in a single elongated cell, called the ascus. As this development is going on, the protoperithecium darkens and forms a flask-shaped structure called the perithecium with an opening, called the ostiole. The mature, dormant ascospores are shot out through the ostiole. The ascospores (haploid cells) germinate when conditions are appropriate and produce a mycelium that contains haploid nuclei that have only one chromosome of each type and therefore one copy of each gene. The fungus displays the alternation of haploid and diploid phases but the haploid phase persists for a long time and is the dominant phase. The haploid mycelium produces the characteristic pink-orange colored conidia by which the fungus is easily recognized in nature and collected.

Approximately 65% of all Neurospora that have been collected globally are comprised of one species called N. intermedia (Turner et al., 2001). Its life history was determined based on observations of the fungus on burnt sugar cane in agricultural fields and from simulated experiments in the laboratory (Pandit and Maheshwari, 1996). Sampling of propagules in air in sugar cane fields did not reveal ascospores, discounting the theory of their aerial dissemination. However, virtually all samples of soil from the sugar cane field, after it had been heated to 60°C for 30 to 45 min—a treatment that kills conidia—yielded Neurospora. In the field-cultivated sugar cane, Neurospora colonies were on the burnt stumps or the stubbles in contact with the soil, suggesting that ascospores present in soil infect the burnt cane. To confirm this, cane segments were planted in soil into which reciprocal mixtures of ascospores and of conidia of wild type (al+, orange) and a color mutant (al, albino/white) had been mixed. The cane segments were burnt in the laboratory, simulating the conditions in the field. The phenotype of Neurospora colonies that developed on the cane segments was that of the genotype of the ascospores that had been mixed into the soil, confirming that the heat-resistant ascospores in soil initiate infection of burnt cane. In nature, the activation of dormant ascospores is brought about by heat generated by burning, or by furfural — a compound produced on heating xylose that occurs as a constituent of the polysaccharide xylan in the plant cell walls.

The life cycle of Neurospora illustrates that most fungi produce spores both asexually and sexually, i.e., fungi are pleomorphic. Environmental conditions determine when, where and how reproduction will occur. On the burnt sugar cane, conidia formed in the sugar-rich cane tissue and their production continued until the sugars had become depleted. The production of astronomical numbers of conidia externally and their constant dissemination by wind is seemingly wasteful but it is the fungus' strategy of removing sugars from plant tissue and to create a nutritional environment for sexual reproduction to occur (Pandit and Maheshwari, 1966). The depleted nutrient conditions favors the development of protoper-ithecia and microconidia, i.e., for sexual reproduction to occur. Further, so long as sugar is available, the absorptive mycelium ramifies inside the tissue and forms closely packed lateral aggregates of conidiophores (sporodochium) beneath the epidermal tissue. The growth pressure of sporodochium causes the epidermis to separate from the ground tissue, thereby creating tissue pockets in which microconidiophores and protoperithecia, a biological solution necessary to ensure humid conditions and attract microfauna for effecting fertilization by transmitting microconidia to trichogyne. The sexual phase is relatively inconspicuous, develops at a different location and may appear unrelated, and was therefore unnoticed for a long time. The occurrence of morphologically distinct asexual and sexual phases at different times due to their differing environmental requirements illustrates that in fungi the condition for asexual and sexual reproduction are often quite different.

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