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TERM PAPER

on
Fungus
Gibberella fujikuroi
Submitted By
SHASHI SHARMA
Roll No.P8003B15
Reg No. 11006142
M.Sc. Micro.

Lovely School of Sciences

Lovely Professional University, Phagwara
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Gibberella fujikuroi (Swada) Wollenw, (1931)

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Whenever a study is conducted, it is done on the basis of certain objective In mind.
A successful completion of a term paper is based on the objective of the study that
could be stated as under:
@m How ?

 
 produces gibberellic acid which is of industrial
importance.
@m Role of gibberellic acid in seed germination,parthenocarpy etc

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?


 is a fungal plant pathogen. It causes

 disease in rice
seedlings, by overloading them with the phytohormone gibberellin as its own
metabolic by product.

The ascomycetous fungus Gibberella fujikuroi, conidial stage F. moniliforme,

causes bakanae, foot rot, seedling rot, grain sterility and discoloration.

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Gibberella fujikuroi is predominately seed-borne; infected kernels may develop a

reddish discoloration due to the presence of the pathogen¶s conidia.

   

Morphological characters observed within Gibberella fujikuroi are

(a) macroconidia; (b) microconidia; (c) microconidia produced in false heads; (d)
microconidia produced in chains; (e) sterile coils.

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Seedlings with bakanae emerge rapidly, remain tender and are significantly taller
than non-infected plants. Severely diseased seedlings die before transplanting, and
those that survive may die after transplanting. Heavily infected seed lots may
produce both, stunted and elongated seedlings. Older infected plants show tall
tillers with pale-green flag leaves well above the canopy of the healthy crop.
Infected plants produce only few tillers and leaves dry up before maturity. In some
cases, diseased plants survive until maturity, but are sterile and produce no or
empty panicles. White powdery growth of conidiophores may be visible on lower
parts of diseased plants. The panicles of healthy plants may be contaminated by
spores and turn pink due to growth and sporulation of the fungus on hulls or
kernels.

 

In the soil as thick-walled hyphae or macroconidia. The fungus produces hyaline,
elliptical ascospores (10 ± 20 x 4 - 7 µm, 1 ± 3 septa), hyaline, oval to clavate,
single-celled microconidia (5 ± 12 x 1.5 ± 2.5 µm) in chains, and hyaline, slightly
curved macroconidia (25 ± 60 x 2.5 ± 4 µm) with a foot-shaped basal cell and 3 ± 7
septa.

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G. fujikuroi produces a variety of biologically active compounds, e.g. fusaric acid
causing stunting, gibberellin causing elongation of many plant species besides rice,
and fumonisin which causes equine leuko-encaphalomalacia and is reported to be
cancerogenic.

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The ascomycetous filamentous fungus ?

  is used to produce

gibberellins,fungal hormones which are used commercially to elevategrowth in
higher plants, including agriculturally andhorticulturally important species. In the
fungus, gibberellinsappear to promote elongation of hyphae and asexualspore
germination.Some strains of the fungus Gibberella fujikuroi (perfect stage of
Fusarium moniliforme) are the industrial source of gibberellic acid.These strains
infect rice and cause the disease known as bakanae in Japan

?

  

@m Role of Gibberellic acid is more or less like auxin. Like auxin gibberellic
acid influence cell division ,cell elongation ,flowering , fruiting ,seed
germination ,cambial activity ,axillary bud formation ,etc.Unlike auxin
,gibberellic acid is transported in all direction.
@m  It increases the number and size of the cells.The inter node
is lengthened under its influence ,as a result the plant becomes long.
@m  Under the influence of gibberellic acid leaves ,flowers and
fruits increase in size.
@m Seedless fruits are formed from it.
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  It removes the dormancy of seeds and influences
the seeds to germinate.
@m    
 Some flowers require light and appropriate
temperature for blooming but under its influence such light and temperature are
not required.
@m  By applying appropriate amount of gibberellic acid the
number of male flowers can be diminished and number of female flowers can
be increased. . Formation of male flowers is generally promoted by
concentrations of 10 to 200 ppm., female flowers by concentrations of 200 to
300 ppm. Concentrations of more than 600 ppm markedly suppresses initiation
of both male and female flowers.
@m ? 
 It inhibits the growth of buds and root.
@m ` ¢warf plants with short inter nodes and rosette of leaves are formed
in winter.These plants in the next year develop elongated inter nodes and bear

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flower,a process called
 Application of gibberellic acid can induce

bolting in winter.
@m ?     Some seeds require light for germination like
lettuce.They do not germinate in darkness.Gibberellic acid can induce
germination in these seeds in complete darkness.
@m  . GA inhibits the formation of roots in cuttings.
@m 
  . Pollination within self-incompatible clones and between
closely related species may some times be forced by the application of GA and
cytokinin to the blooms at the time of hand pollination.

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Gibberellins of commercial value, GA,, GA, and GA,are used in combinations as it

is uneconomic to separate these molecules onan industrial scale.
@m   Gibberellic acid increases size of table grapes.
@m   Application of gibberellic acid increases the amount of
-amylase in germinating barley.This enzyme is used in the production of malt
for beer industry.
@m     Gibberellic acid can prevent quick ripening of
fruits.Because post harvested crops decay quickly due to senescence,so if the
seeds of apple ,orange are spread with a solution of gibberellic acid ,their early
senescence can be prevented.

  

Geberellic acid must be carefully calibrated. When too much is applied, the results
may be the opposite of those desired. If too little is applied, repeat treatments may
be required. The acid is approved by most organic certification programs, because
it is found naturally in plants.

Many of the table grapes grown in the United States are a genetically seedless
variety that would naturally produce small fruit on very compact clusters. Almost
all seedless grapes on the market are treated with GA3. It substitutes for the
presence of seeds, which would normally be the source of native GAs for fruit

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growth. Repeated spraying with GA3 increases both rachis length (producing
looser clusters) and fruit size.The increased rachis length prevents the cluster from
being too compact, and this reduces the chance of fungal growth inside the cluster.
Two to three additional applications of GA3 during fruit development are thought
to increase berry size by enhancing the import of carbohydrates into the developing
fruit. In excess of 8 tons of GA3 are used in the California grape industry annually.

Gibberellin induces growth in Thompsons seedless grapes. The bunch

on the left is an untreated control. The bunch on the right was sprayed
with GA3 during fruit development.

Gibberellic acid is also used to boost cherry production. Sweet, bing cherries are
sprayed 4 to 6 weeks before harvest to increase fruit size. Application of GA3 to
tart cherries increases yield through enhanced bearing.

Gibberellin A4 (GA4) is used to promote the fruit set of apple and pear trees. For
example, in some apple cultivars the amount of fruit produced is often limited by
biennial bearing, a phenomenon whereby the production of a heavy crop of fruit
one year inhibits the subsequent production of flower buds, and hence, the yield of
fruit the following year.The alternate bearing of some cultivars can be overcome
by applying GA4 in the "off" year to promote the formation of flower buds, and
subsequent fruit set. In regions of Europe where fruit set of apple and pear trees is
often reduced by inclement weather at the time of pollination, the application of a

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hormone mixture can promote the production and subsequent growth of

parthenocarpic (seedless) fruit. GA4/7 is also used on Golden ¢elicious apples to
prevent abnormal cell divisions in the epidermal layer that produce "russetting."
The russetted appearance of the apple fruit is considered by many consumers to be
undesirable.

Gibberellic acid is also applied to citrus crops, though the actual use depends on
the particular crop. For example GA3 is sprayed onto oranges and tangerines to
delay or prevent rind-aging, so that fruit can be harvested later without adverse
effects on rind quality and appearance. For lemons and limes, GA3 synchronizes
ripening and enhances fruit size.

Gibberellic acid is used extensively to increase the sucrose yield of sugarcane.

Sugarcane, a normally fast-growing C4 member of the Poaceae (grass) family, is
sensitive to cooler winter temperatures, which reduce internode elongation and
subsequent sucrose yield. The adverse effects of cooler temperatures can be
counteracted by the application of GA3.

GA-induced Į-amylase synthesis in the aleurone of cereal grains. Gibberellins

from the embryo of germinating grains are necessary for the synthesis of Į-
amylase by the cells of the aleurone layer, which, in turn is necessary for the
hydrolysis of starch within the endosperm.In the brewing industry, the production
of beer relies on this hydrolytic breakdown of starch in barley grains to yield
fermentable sugars, principally maltose, which are then subjected to fermentation
by yeast. ¢uring fermentation, glycolytic enzymes from yeast break down the
sugars, resulting in ethanol.

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Structure of a germinating barley grain and the biochemical processes that occur
during the "modification" part of the malting process.

In the multistep malting process, mature barley grains are steeped or soaked to
allow them to imbibe water. Next, the grains are spread out to germinate, during
which time the starch within the endosperm will be hydrolyzed by Į-amylase
allowing the embryo to begin to grow. This process of starch breakdown is referred
to as "modification." Gibberellic acid may be applied during this time and will
supplement the native GAs in the grain, enhance the production of Į-amylase, and
consequently, speed up the hydrolysis of starch. (The molecular aspects of this GA
response are shown in Chapter Figure 20.24). The germinated grains, which show
a well-developed root and have a shoot (termed an "acrospire"), which is
approximately 75% of the length of the grain, are then kilned. This is a two-step
process, first to dry the grain, and second, to cure or heat it. The temperature to
which the modified malt is cured will determine whether light-colored (low-
temperature curing) or dark beer (high-temperature curing) is to be produced. The
modified and kilned malt provides the raw material for the fermentation.

  
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Gibberellins were discovered by Japanese plant pathologists studying "bakanae"

disease ("foolish seedling") of rice, in which seedlings grow elongated and die. In
1898 Shotaro Hori demonstrated that it was caused by a fungus, now known as
?


 . In 1926 Eiichi Kurosawa reported that a chemical produced
by the fungus caused the symptoms, and that the substance was heat-resistant, not

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losing its activity after 4 hours at 100°C (212°F). In 1935 Teijiro Yabuta first
isolated a non-crystalline solid and named it Gibberellin. In 1938, Yabuta and
Yusuke Sumiki first isolated a crystalline compound from the cultured fungus.

Since this time, 79 different gibberellins have been isolated, many of these from
the seeds of a wide variety of species. Gibberellic acid-3 (GA-3) is the most widely
used, and is produced commercially by growing the fungus in huge vats and then
extracting and purifying the GA-3.

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  (also called ?

 , ?, and (?) is a hormone found
in plants. Its chemical formula is C19H22O6. When purified, it is a white-to-pale-
yellow solid.

Gibberellic Acid-3 (GA-3) is a naturally occurring plant growth regulator which

may cause a variety of effects including the stimulation of seed germination in
some cases. GA-3 occurs naturally in the seeds of many species and is produced
commercially by growing ?


 fungus cultures in vats, then
extracting and purifying the GA-3. Presoaking seeds in GA-3 solution will in many
cases cause the rapid germination of many types of highly dormant seeds which
would otherwise need cold treatment, after-ripening or aging, or other prolonged
pre treatments. Many different types of dormancy are overcome with GA-3, and
there are excellent results with many ordinarily difficult seeds, including some
types which have never before succeeded with. Not all seeds respond well. A great
deal of research needs to be done to determine which species benefit, and the
proper concentration of GA-3 for each type.

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GA-3 is a   plant growth regulator. It is a completely natural,

organic substance that is present in many plants, including many common foods,
and in fact is essential to certain life-processes in many plants. There is absolutely,
positively,   about it. It is produced by growing a naturally-
occurring fungus in large vats and extracting the GA-3 from it. It is NOT produced
synthetically by any chemical process, but is EXTRACTE¢ from a plant (fungus),
so it is just like many vitamins which are extracted from plants, or penicillin which
is extracted from fungus. Its chemical structure is not changed in any way. Yes, it
is sold under the chemical name, so it SOUN¢S "chemical" but is no less natural
than the vitamin C that is extracted into the water of a cup of rose-hip tea, or the
vitamin E extracted from wheat germ.

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Gibberellins are formed from acetyl-CoA which is first converted to mevalonate

pyrophosphate takes part in further synthesis.It is changed first to geranyl
pyrophosphate and then trans-geranylgeranyl pyrophosphate.Copalyl
pyrophosphate is the first cyclic compound.It gives rise to

kaurene and its various derivatives.They are precursors of gibberellins but the
exact pathway is yet to be elucidated.

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GA-3 occurs naturally in the seeds of many species and is produced commercially
by growing ?


 fungus cultures in vats, then extracting and
purifying the GA-3. More than 4 tons of gibberellins are sold annually.

In aerobic fermentations involving fungi, their filamentous nature commonly leads

to excessive viscosity in the fermentation broth and demands higher agitation and
aeration to maintain satisfactory levels of dissolved oxygen (¢O). The expenditure
on energy for aeration and agitation of such viscous broths is considerably high. It
is a common observation that G. fujikuroi also grows in viscous, filamentous form
in liquid medium and hence its submerged cultures often become oxygen limited.
Rate of growth of G. fujikuroi and production of GA3 in fermenter is governed to a
considerable extent by oxygen transfer in the fermenter. It is also reported that G.
fujikuroi cultures enter a linear growth phase after initial logarithmic phase

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(Borrow et al. 1964) presumably because of the oxygen limitation. Any change in
morphology of the fungal strain that lowers the viscosity can result in improved
oxygen transfer and in turn, increase the GA3 production.

¢uring stationary phase of G. fujikuroi culture several products like bikaverin,

carotenoids, gibberellic acid, sterols and lipids are produced from a common
precursor, acetyl-CoA. Their concentrations and ratio are governed by availability
of oxygen in the culture (Giordano and ¢omenech 1999). A decrease in production
of pigments like bikaverin and carotenoids by G. fujikuroi is likely to be beneficial
for production of gibberellins because of the increased carbon flow through the
gibberellin pathway as well as requirement of lesser steps during extraction and
purification of the gibberellins from fermented broth.

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Gibberellin production by Gibberella depends upon the nature of the carbon and
nitrogen sources and is stimulated by a high carbon to nitrogen ratio.Gibberellin
production follows cessation of growth and exhaustion of the nitrogen source in
batch cultures and does not occur at low growth rates and low nitrogen
concentrations (less than 65 mg L`'in the form of glycine) in a chemostat. The
onset of gibberellin production was attributed to the growth arrest that follows the
depletion of an essential nutrient .This is usually assumed to be a general trait of
secondary metabolites.

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Gibberellin production by Gibberella fujikuroi started only after the nitrogen
source was depleted and ceased upon its renewal.Nitrogen repression of gibberellin
biosynthesis is not an indirect effect of the growth arrest that follows the depletion
of an essential nutrient because gibberellins were not produced upon depletion of
phosphate. Mycelia produced gibberellins when suspended in a glucose solution.
Production ceased some time after depletion of glucose and resumed upon its
readdition. Under certain conditions, the gibberellin production rate was inversely
proportional to the glucose concentrations.The specific regulation of gibberellin
biosynthesis by the nitrogen source imposes a revision of the concept that
gibberellins are secondary metabolites whose production is triggered by imbalance
or cessation of growth.


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The carbons in gibberellins must evidently come from a carbon source. It is thus
not surprising that carbon limitation did not allow gibberellin production. When
the nitrogen concentrations were large enough to allow glucose to be exhausted
first, no gibberellins were produced.In low-nitrogen, low-glucose media,gibberellin
production stopped after a few days. Addition of glucose was sufficient for
gibberellin synthesis to resume at a constant rate.This rate represented a sort of
'installed capacity' for gibberellin production; when the initial glucose and nitrogen
supplies were halved, the rate was halved.A culture medium was not required for
gibberellin production by previously uninduced mycelia: transfer of mycelium
grown in high-nitrogen minimal medium to a glucose-phosphate solution led to
lasting gibberellin production.The activation of gibberellin production upon
transfer was not caused by transfer stress, because it did not occur in mycelia
returned to the previous medium.In experiment, transfer to a glucose solution gave
the the same results as transfer to a glucose-phosphate solution. Therefore,
phosphate was not required for gibberellin production.Glucose inhibited
gibberellin production. High initial glucose concentrations were deleterious for the
accumulation of gibberellins.The productivity of young mycelia(aged 3-6 d) was
very negatively related to the glucose concentrations present in the media at the
time.

Therefore,Gibberellin production occurs only after depletion of the nitrogen source

and is inhibited when this source is renewed. This phenomenon ia called as
nitrogen repression,without implying any conclusion as to the underlying
mechanism at the level of gene expression or enzyme activity.Nitrogen repression
is not an indirect consequence of the imbalance or the cessation of growth that
accompanies depletion of an essential nutrient: gibberellin production was not
induced by phosphate depletion in the presence of excess glucose and nitrogen
sources. Nitrogen repression is reversible;gibberellins are produced following
removal of the nitrogen
source from nonproducing cultures..The appreciable gibberellin synthesis in the
presence of high-nitrogen concentrations suggests that their strain, a high
gibberellin producer, is partially insensitive to nitrogen repression.The absence of
nitrogen is no obstacle for the production of gibberellins because these molecules
contain no nitrogen atoms. Gibberellins are synthesized for many days by mycelia
for a remarkably long time, at least 6 d, after depletion of glucose. The requirement
of a carbon source in gibberellins production contrasts with the observation of a
strong antagonism of glucose concentration and the gibberellin production rate. A
gradual dispensation of the carbon source improves gibberellin

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production.Nitrogen repression and the negative effect of high-glucose

concentrations should reflect the conditions under which
gibberellins are produced in the course of the infection of rice plants by
Gibberella.A wealth of nitrogen and carbon sources in a plant should delay the
onset of gibberellins production by the fungus to a later stage in plant
development.

 

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 produce gibberellin acid.Gibberellic acid is used in grape
industry,brewing industry and in agriculture.This acid has a role in stem
elongation,Parthenogenesis,breaking of dormancy,increase in size of
fruit,production of malt and has antisenscence activity.so because of these uses
this fungus has huge scope in today¶s world. Gibberellins are the only ones
produced through fungal fermentation. A total of 79 gibberellins are currently
known; 25 of which are produced by fungi. Gibberellins were first isolated from
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 , the sexual state of    , when it was
discovered that rice infected with this fungus grew faster than normal. It¶s
importance as a plant growth stimulator was soon recognized and more than 4
tons of gibberellins are sold annually. ?


 and  
   are used industrially where fermentation of substrates is done in 5-
6000 gal vats.

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Curtis PJ, Cross BE. 1954. Gibberellic acid. A new metabolite from the culture
filtrates of Gibberella fujikuroi. Chem. Ind. 1066.

Grove JF. 1961. The gibberellins. Quart. rev (Chem. Soc. London) 15, 46-70.

Hori S. 1898. Some observations on "Bakanae" disease of the rice plant. Mem.
Agric. Res. Sta. (Tokyo) 12 (1),110-119.

Kurosawa E. 1926.Experimental studies on the nature of the substance secreted by

the "bakanae" fungus. Nat HistSoc Formosa. 16, 213-227.

Macmillan J, Suter PJ. 1958. The occurence of gibberellin A1 in higher plants:

Isolation from the seed of runner bean (Phaseolus multiflorus). Naturwiss 45, 46.

Macmillan J, Seaton JC, Suter PJ. 1959. A new plant-growth promoting acid-
gibberellin A5 from the seed of Phaseolus multiflorus. Proc. Chem. Soc. 325.

Macmillan J, Seaton JC, Suter PJ. 1960. Isolation of gibberellin A1 and gibberellin
A5 from< i>Phaseolus multiflorus. Tetrahedron. 11, 60-66.

Macmillan J, Seaton JC, Suter PJ. 1962. Isolation and structures of gibberellin A6
and gibberellin A8. Tetrahedron. 18, 349-355.

MacMillan J, Takahashi N. 1968. Proposed procedure for the allocation of trivial

names to the gibberellins. Nature. 217. 170-171.

Mitchell JE, Angel CR. 1951.The growth-stimulating properties of a metabolic

product of Fusarium moniliforme.Phytopath. 41,26-27.

Phinney BO. 1983 The history of gibberellins. In: The Biochemistry and
Physiology of Gibberellins (Ed. Crozier A.) vol 1. pp 19-52. Praeger Publishers
USA.

Phinney BO, West CA, Ritzel MB, Neely PM. 1957. Evidence for gibberellin-like
substances from flowering plants. Proc. Nat. Acad. Sci. (U.S.A.) 43. 398-404

Radley M. 1956. Occurrence of substances similar to gibberellic acid in higher

plants. Nature 178. 1070-1071

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Stodola FH, Raper KB, Fennell ¢I, Conway HF, Johns VE, Langford CT, Jackson
RW. 1955. The microbial production of gibberellins A and X. Arch. Biochem.
Biophys. 54, 240-245.

Takahashi N, Kitamura H, Kawarada A, Seta Y, Takai M, Tamura S, Sumiki Y.

1955. Isolation of gibberellins and their properties. Bull Agric Chem Soc Japan.
19. 267-277.

Takahashi N, Seta Y, Kitamura H, Sumiki Y. 1957. A new gibberellin, gibberellin

A4. Bull Agric Chem Soc Japan. 21. 396-398.

Tamura, S. 1990. Historical aspects of gibberellins. In: Gibberellins ( Eds

Takahashi N, Phinney BO and Macmillan J) pp 1-8,Springer-Verlag New York.

Yabuta T, Sumiki Y. 1938. On the crystal of gibberellin, a substance to promote

plant growth. J Agric Chem Soc Japan. 14. 1526.