`
`THE INTERNATIONAL RESEARCH GROUP ON WOOD PRESERVATION
`
`Section 3
`
`WOOD PROTECTING CHEMICALS
`
`Borates and their biological applications
`
`JD Lloyd
`
`Borax Europe Limited, Priestley Road, Guildford, GU2 5RQ, United Kingdom
`
`Paper prepared for 29th Annual Meeting
`Maastricht, Netherlands
`14-19 June 1998
`
`IRG Secretariat
`S-100 44 Stockholm
`Sweden
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`Borates and their Biological Applications
`
`JD Lloyd
`Borax Europe Limited, 170 Priestley Road, Guildford, GU2 5RQ, United Kingdom
`
`Abstract
`This paper reviews some of the many biological applications of borates. Boron is a
`ubiquitous element found widely distributed in the environment and is a normal
`component of a healthy diet. Elemental boron does not exist in nature, but is always
`found combined with oxygen in compounds called borates. Boron is an essential micro(cid:173)
`nutrient for plants, and there is evidence to suggest that boron is of nutritional
`importance, if not essential, for humans. Borates possess biostatic activity which
`enabled their use in medicine and has allowed their continued development as
`preservatives.
`
`The essentiality of boron in plants has led to extensive biological use in agriculture.
`The biostatic properties at high doses have enabled their use in biodeterioration control,
`against insects, fungi, algae and bacteria. Some use is currently being made of borates
`for insect control in the home.
`
`The application of borates to crops, to alleviate boron deficiency, has resulted in
`recognized increases in quality and yield. Consideration of the relative safety and
`effectiveness of borates as biocides, is expected to lead to an increase in the use of these
`products in the future.
`
`Key Words: Applications; Biochemistry; Biology; Borates; Boron; Chemistry;
`Micronutrient; Preservative.
`
`Introduction
`The objective of this paper is to review some of the large scale commercial applications
`of boron which interact with biological systems. The relevant chemistry and
`biochemical interactions, which render borates bioactive molecules, are introduced.
`Boron chemistry and biochemistry is of particular interest as all of its physiological
`effects are as a direct result, even though some appear at first to be contradictory.
`
`The two most well known which will be discussed in detail here are the essentiality of
`borates on the one hand and their toxicity or effectiveness as a preservative on the other.
`This is perhaps not such a surprise when one considers the ubiquitous nature of borates
`and the ability of most micro-nutrients to become detrimental at high physiological
`concentrations.
`
`Boron is a trivalent element widely distributed in the environment, comprising about
`0.001 % of the earth's crust (The Merck Index, 1989); concentrations average 3-10 µgig
`in soil (Adams, 1964; Muetterties, 1967), 4.5 µg/g in ocean waters (Weast, 1983), and
`about 0.01 µg/g in freshwater (Jenkins, 1980).
`
`Boron is widely distributed in plant and animal tissues and is known to be essential for
`plant growth (Gouch & Dugger, 1954; Skok, 1958; Skol'nik, 1974; Underwood, 1977;
`Lovatt & Dugger, 1984). In a review on its toxicity by Murray (1995), it was reported
`the average U.S. daily dietary intake of boron is 1.5 mg B/day. The median
`that:
`boron content of U.S. drinking water
`is 0.031 µgig with a maximum of 3.95 µg/g
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`(EPA, 1994) and the daily boron intake for humans from food and water has been
`estimated at 0.5 - 3.1 mg for adults (Nielson, 1992).
`
`Chemistry of Boron
`Boron is the only non-metal in a family otherwise comprised of active metals, group
`lllb of the periodic table. As could be expected, boron exhibits bonding and structural
`characteristics intermediate to both, as do other elements lying to either side of the
`metal/non-metal border. Boron (Atomic number 5) also has a tendency to form double
`bonds and macromolecules, although these bonds are more correctly described as partial
`double bonds and are due to 7t electron back bonding into the empty p orbital of boron.
`
`Because of an incomplete electron octet, boron compounds can act as electron pair
`acceptors and this behavior is demonstrated by the Lewis acid properties of boron. It is
`this tendency which is fundamental when forming hypotheses attempting to predict the
`action of boron within biological systems, as will be discussed.
`
`Boron does not occur in nature in its elemental form, but rather as oxygen containing
`compounds such as boric acid (B[OH]3), in some volcanic spring waters and elsewhere,
`as borates such as borax. These compounds are used as commercial products and for the
`synthesis of other boron compounds. In this paper and elsewhere, references to boron in
`the environment and in various applications, have referred to the elemental boron
`content, which in some cases allows for comparisons between studies and applications.
`
`Oxygen containing compounds of boron are among the most important, comprising
`nearly all the naturally occurring forms. The structures of these compounds consist
`mainly of trigonal BO3 units with sp2 hybridization, and with tetrahedral BO4 units
`
`with sp3 hybridization (Cotton et al., 1987). B-O bond energies are 560 - 790 kJ, with
`the only competition in strength offered by the B-F bond in BF3 (640 kJ) (Cotton &
`Wilkinson, 1986). Endless organic derivatives containing boron-oxygen bonds are
`known; the main examples that include trigonal boron are the orthoborates (B[OR]3 e.g.
`esters; the acyl borates (B[OCOR]3); the peroxo borates (B[OOR]3; and the boronic
`acids (RB[OH]2). It is appropriate to consider that these are derivatives of boric acid
`(Cotton et al., 1987).
`
`Boric acid is a as colorless, odorless, transparent crystals or as white granular powder
`(Anon, 1980). It is readily soluble in water, ethanol and glycerol (Merck Index, 1989).
`Borax is a white crystalline substance and is soluble in water and glycerol, but insoluble
`in alcohol (Merck Index, 1989). Three other sodium borates are commonly known:
`sodium metaborate (NaBO2); sodium perborate (NaBO3 [4H2O]); and sodium
`
`pentaborate (Na2B10O16· 10H2O). Like boric acid and borax, they are soluble in water
`and glycerol (ibid).
`
`It is these oxygen containing compounds of boron that are traditionally used in
`biological applications. Boric acid, borax, mixtures of the two or a spray dried mixture
`equating roughly to disodium octaborate tetrahydrate, being the most commonly used,
`although organic esters of boric acid such as trihexylene glycol biborate, are also
`currently in use and boronic acids are used in minor applications. Perborates, boric acid
`and borates, have been used in the past as general antiseptics or bacteriostats (Anon.,
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`1980; Merck, 1989) and in fact boric acid was used as an antiseptic by Sir Joseph Lister,
`the father of modem surgery, in the mid 19th century.
`
`Boric acid is moderately soluble in water, but has a large negative heat of solution so
`It is a very weak and
`that the solubility increases markedly with temperature.
`exclusively monobasic acid that is believed to act, not as a proton donor, but as a Lewis
`acid, accepting OH-:
`
`========'~
`B(OH)3 + H20 '-
`B(OH)4- + H+
`
`pK=9.00
`
`The B(OH)4- ion actually occurs in some minerals. At concentrations less than
`
`0.025M, only the mononuclear species B(OH)3 and B(OH)4- exist; but at higher
`concentrations the pH becomes consistent with the formation of polymeric species such
`as:
`
`3B(OH)3 '-
`
`::::,,,
`
`pK=6.84
`
`It is also likely that polymers exist in mixed solutions of boric acid and borates, such as:
`
`;;::======'~
`2B(OH)3 + B(OH)4- '-
`
`Boron (in the form of boric acid or more probably ionized as the tetrahydroxy borate
`anion), is one of the chemical elements whose oxygen compounds will form chelate
`complexes with certain organic compounds containing cis adjacent alcohol groups:
`
`HO,
`R
`HO/
`
`/0, ~OH
`== R
`B
`'o/ ',oH
`
`and
`
`✓ o"n,oH +
`"-.o/ '',OH
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`These complexes occur in aqueous solution and are well known. Biot as early as 1842,
`reported that a solution of boric acid became acidic to litmus upon the addition of sugars
`and Thompson (1893) found that boric acid could be determined by titration in the
`presence of various polyhydroxy compounds (or polyols).
`
`Many chemical and most biochemical reactions of boron are based on the reactivity of
`the borate anion with adjacent alcohol groups. These stable complexes formed by rapid
`esterification with polyols (Nickerson, 1970; Boeseken, 1949), are mainly 1 : 1 and
`charged. It has also been suggested that similar reactions take place with a -hydroxy
`carboxylic acids (Kustin & Pizer, 1969) and although this is true of gluconate for
`example, it has been shown that the complex is only formed as a result of an additional
`hydroxyl group in the p position as other a -hydroxy carboxylic acids such as lactate do
`not undergo the same reaction (Lloyd, 1993). The type of complexes formed with
`polyols depends on pH and on the ratio of the borate ion to the diol (Zittle, 1951) and
`the acidity of the hydroxy groups. When a low diol to borate ratio exists, it has been
`proposed that the monoester is prevalent, whilst when the diol to borate ratio is high,
`then the diester is predominant. The acidity of boric acid is thereby increased as
`mentioned above. Steric considerations are critical in the formation of these complexes.
`Thus 1,2- and 1,3-diols in the cis-form only, such as cis-1,2-cyclopentanediol are active,
`and only o-quinols react. Indeed the ability of a diol to affect the acidity of boric acid is
`a useful criterion of the configuration where cis-trans-isomers are possible.
`
`This specific complex forming ability has led to the use of boric acid in carbohydrate
`separation and in determining carbohydrate configuration (Boeseken, 1949; Annison et
`al., 1951; Khym & Zill, 1951; 1952; Popiel, 1961).
`
`Biochemical Effects of Boron
`Several compounds of biological importance such as vitamins and co-enzymes can react
`to form complexes with the borate ion (Zittle, 1951; Aruga, 1985). Reactions with these
`molecules and others within the cell, have been found to produce dramatic changes in
`metabolism.
`
`A good example of this is the effect of boron on plant metabolism and lignification.
`Here the borate seems to play at least one role by partitioning metabolism between the
`pentose phosphate shunt and glycolytic pathways. Boron deficiency has been shown to
`result in an accumulation of phenolic compounds in plants (Dugger, 1983; Shkol'nik,
`1974). An increase in lignification has also been observed under these conditions
`(Acerbo et al., 1973). Such metabolic changes appear to be caused by a lack of the
`inhibition of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, which are
`normally inhibited in the presence of boron. This inhibition
`in the case of 6-
`phosphogluconate dehydrogenase was suggested to result from the formation of a
`complex with boric acid and the a-hydroxy carboxylic acid, 6-phosphogluconate (Lee
`& Aronoff, 1967) but has been subsequently shown to be as a result of chelate formation
`with the co-enzyme NADP+ which also features in the reaction (Lloyd et al., 1990;
`Lloyd & Dickinson, 1991; Lloyd, 1993).
`In the absence of boron, the pentose
`phosphate pathway is left unregulated and results in an over-production of phenolic
`acids and other lignin components. The accumulation of such compounds would not
`only lead to the necrosis of plant tissue, but also to an increased deficiency problem, as
`some of these phenolics, also having alcohol groups, would complex with boric acid
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`(Shorrocks, 1990). In the presence of boron, the inhibition of glucose-6-phosphate and
`6-phosphogluconate dehydrogenase will restrict both the flux of substrate into the
`pentose phosphate pathway and the synthesis of phenols. As a result of this, glycolysis
`and the synthesis, for example of hemicellulose and related cell wall material will
`mcrease.
`
`Frost (1942) found that borate forms complexes with the ribityl group of riboflavin and
`this was then shown to inhibit the activity of the flavoprotein xanthin oxidase (Roush &
`Norris, 1950). Because of the borate ion complexing with the ribityl group of another
`flavin containing molecule FAD+ (flavin adenine dinucleotide) (Shepherd, 1951) the
`effect of borate on NAD+ (nicotinamide adenine dinucleotide) requiring enzymes was
`investigated: borate inhibited yeast alcohol dehydrogenase (Roush & Gowdy, 1961;
`Weser, 1968; Smith and
`Johnson, 1976); yeast glyceraldehyde-3-phosphate
`dehydrogenase (Missawa et al., 1966); and aldehyde dehydrogenase (Deitrich, 1967).
`Evidence given by some workers suggested that the inhibition was due to complex
`formation. This hypothesis was suggested as a result of the removal of inhibition by the
`addition of non-substrate polyols (Roush & Norris, 1950; Roush & Gowdy, 1961 ). It
`was later shown that these inhibitory effects were due to the formation of a complex
`with the ribityl group of NAD+ (Smith & Johnson, 1976). Johnson & Smith (1976)
`showed
`that
`the
`same complexation occurred with NMN+
`(nicotinamide
`mononucleotide), that the reaction took place with the cis-adjacent hydroxyls within the
`ribityl group of NMN+ and NAD+, and that the ribose next to the positively charged
`nicotinamide moiety in NAD+ was preferred (NAD+ has two ribose units). Another
`important flavoprotein that was found to be effected by borate is cytochrome b5
`It was found that
`reductase (NADH-cytochrome b5 reductase) (Strittmatter, 1964).
`borate again interacted with the nucleotide substrate. It was also shown that the addition
`of borate buffers to reduced flavoprotein-pyridine nucleotide complexes results in
`proton transfer from the flavoprotein to the pyridine nucleotide, and release of the
`nucleotide presumably as the borate complex. It could be conceived that this sort of
`borate-cytochrome interaction could result in a partial blocking of the electron transport
`chain.
`
`Other vitamins and co-enzymes found to react with the borate ion include: AD(5)P
`(muscle adenylate); pyridoxine (vitamin B6); dehydroascorbic acid (the reversible
`oxidation product of vitamin C); co-enzyme A; 5-deoxyadenosylcobalamin (vitamin
`B12) and pantothenate (Zittle, 1951).
`
`In addition, borate appears to be able to act as an inhibitor in a purely ionic manner as
`shown in the case of alkaline phosphatase (monoesterase) (Cram & Rossiter, 1949;
`Zittle & Della Monica, 1949). The phosphodiesterase is inhibited by borate too,
`although this may be due to an interaction with enzyme bound polysaccharide.
`
`Another slightly different form of inhibition is seen in the case of serine-acyl-enzymes.
`These enzymes, the most noted of which being the serine proteases, contain an
`unusually reactive serine residue ( e.g. serine 195 in chymotrypsin) at the active site of
`the enzyme. These enzymes have been shown to be inhibited by the formation of a
`transition state inhibitor, via complex formation between the hydroxyl group of the
`serine residue and boric, or boronic acids. The close proximity of a cationic histidine
`residue may also be of importance. The reactive serine residue can be labeled quite
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`specifically by the formation of an inactive diisopropylphosphoryl/enzyme complex by
`the addition of the organic fluorophosphate diisopropylphosphofluoridate. Other serine
`enzymes that are deactivated by diisopropylphosphofluoridate have also been shown to
`be inhibited by boric and boronic acids (Berezin et al., 1967; Bauer & Pattersson, 1974;
`Lindquist & Terry, 1974; Mathews et al., 1975), including choline esterase (Garner &
`Pelly, 1984). Another serine class of enzyme is phosphoglucomutase, which plays a
`role in glucose metabolism. This is a phospho-enzyme rather than an acyl-enzyme, but
`still uses the active serine residue at the center of the active cite. As could be expected
`this enzyme has also been shown to be inhibited by borates (Loughman, 1961; Parr &
`Loughman, 1983).
`
`It is also of interest that enzymes using serine as a substrate can also be inhibited with
`boric acid by the formation of a transition serine/borate/enzyme complex as with y(cid:173)
`glutamyl transpeptidase (Tate & Meister, 1978). Transpeptidase enzymes are also of the
`serine class, and this interaction is probably an enzyme-serine/borate/serine complex.
`Related to this perhaps is the inhibition of P-lactamases (Amicosante et al., 1989).
`These enzymes attack P-lactams such as penicillin, whose target enzyme happens to be
`the transpeptidase used for cross-linking the peptidoglycan cell walls of some bacteria.
`P-lactamases are therefore likely to have a similar active site to transpeptidases, making
`them accessible to inhibition by boric and boronic acids in a similar way. A number of
`other enzyme systems not discussed in this review have been shown to be inhibited by
`borates. Some of these are listed in table I.
`
`TABLE I Some Additional Enzyme Systems Inhibited By Borates
`
`Enzyme System
`Asparagine semi-aldehyde
`dehydrogenase
`Fructose bisphosphatase
`Hexokinase
`Lecithinase
`EnzymeQ
`Sorbitol dehydrogenase
`Tyrosine hydroxylase
`UDPG pyophosphorylase
`UDPG starch glucosyltransferase
`
`Reference
`Hegemann et al. (1970).
`
`Vergnano et al. (1960).
`Wiebelhaus & Lardy (1949)
`Arnaudi & Novati (1957).
`Gilbert & Swallow (1949).
`Wolff (1955).
`Quick & Sourks (1974).
`Griffith et al. (1978).
`Augsten & Eichhorn (1976).
`
`Boron compounds may also be able to interact directly with hydroxyl rich compounds in
`cellular membranes. Any such interaction could result in a change of functional
`It has been reported that roots and apical meristems grown under boron(cid:173)
`activity.
`deficient conditions contained lower than normal amounts of phospholipids and cell
`structural organization (Shkol'nik & Kopman, 1970). Membrane bound enzyme
`activities in plants have also been shown to be altered as a result of changes m
`membrane permeability induced by boron deficiency (Dave & Kannan, 1980).
`
`Because of the sensitivity of membranes and membrane permeability, to treatments
`such as low calcium or low temperature, many workers have investigated the role of
`boron in plants with respect to membrane permeability (Robertson & Loughman, 1974;
`Pollard et al., 1977; Parr & Loughman, 1983; Dugger, 1983). Artificial membrane
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`systems (liposomes) have been used by Parr & Loughman (1983), to demonstrate a
`direct effect of borate on membrane permeability, and they suggest that if boron in
`plants does contribute to the integrity of the cell membrane, then this could well be its
`primary role in plants. Direct membrane effects have also been shown in fungal
`systems using yeast protoplasts, although concentrations required to observe changes
`were above normal physiological concentrations (Lloyd, 1993).
`
`Boron Essentiality
`The biochemistry of boron in the metabolism of vascular plants encompasses a history
`of about 80 years of research trying to elucidate its primary role. A large proportion of
`this work that has been carried out since 1910, when it was first suggested that boron
`was required for plant growth, has been concerned with showing the boron requirement
`of a variety of plants for both growth and developmental processes (Hewitt, 1963 ). The
`majority, however, has been concerned with trying to determine its primary function in
`metabolism.
`
`Boron in the form of boric acid or borates is an essential mineral element for all vascular
`plants (Gouch & Dugger, 1954; Skok, 1958; Shkol'nik, 1974; Underwood, 1977; Parr &
`Loughman, 1983; Dugger, 1983; Lovatt & Dugger, 1984) and diatoms (Lewin, 1966a,
`b). The essential nature of boron in plants was apparently derived through evolution (Mc
`Clendon, 1976) and may be due to adaptation to its presence. Neither fungi nor fresh
`water algae, however, seem to have a measurable boron requirement, although some
`evidence exists for a role of boron in other organisms, especially those that fix nitrogen
`(Anderson & Jordan, 1961; Gerloff, 1968). Growth of some other organisms has been
`stimulated by the presence of boron, although it did not appear to be essential (Davis et
`al., 1928; Mcilrath & Skok, 1958). Schwarz (1974) suggests that because boron is
`ubiquitous in animal tissues and possesses properties expected of an essential element, it
`should be classified 'under special consideration' for trace element function. Other
`evidence has also suggested that boron has importance in human nutrition, and it has
`recently been considered to be a "probably essential" trace element (WHO, 1996).
`(Ref: WHO, 1996. "Trace Elements in Human Nutrition and Health, WHO, pp. 161,
`17 5-178, 1996) Further information on the importance of boron in animal and human
`diets has been reported by Hunt (1994) and Nielsen (1994).
`
`The essential nature of any element for plants can be established according to a set of
`defined criteria (Amon & Stout, 1939):
`1. the element must be essential to the completion of the life cycle;
`2. the element cannot be substituted for or replaced by any other element;
`3. the element must have a distinct function.
`The first two criteria were fully demonstrated in plants by Sommer & Lipman (1926)
`and Warington (1923) respectively, however, the final criteria has yet to be proven,
`although it is generally accepted that boron is an essential element in plants.
`
`As mentioned previously, boric acid and borates are able to form complexes with
`compounds containing certain configurations of alcohol groups. Most hypotheses that
`attempt to explain the role of boron in plants are based upon this reaction and these have
`been reviewed by Shorrocks (1990). A substantial proportion of the boron content in
`higher plants seems to be complexed as stable cis-borate esters in the cell walls (Thellier
`et al., 1979). The fact that the boron requirement of dicotyledons is higher than
`monocotyledons, is presumably related to the higher proportions of compounds with
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`cis-diol configuration in the cell walls, mainly in the hemicellulose fraction and in lignin
`precursors (Lewis, 1980). It has been suggested that the function of this apoplastic
`boron is somewhat similar to that of calcium, in both regulating synthesis and
`stabilizing certain cell wall constituents, including the plasma membrane (Shorrocks,
`1990). According to Lewis (1980) the functions of boron are primarily extracellular
`and, where intracellular are related to lignification.
`
`Metabolic regulation by boron is hypothesized in plants, again by virtue of its ability to
`form complexes with polyols. When the borate ion complexes with compounds that are
`reactants or products of enzymatic reactions in plants, it may stimulate or inhibit the
`course of specific metabolic pathways. The effect of boron on the pentose phosphate
`pathway (Lee & Aronoff, 1967), production of phenolics and lignification (Lewis, 1980;
`Shorrocks, 1990) is a good example of this. These altered pathways may in turn bring
`about altered metabolite pool sizes, which in their turn can bring about altered plant
`growth or development. In a review on boron in plant metabolism (Dugger, 1983) a
`hypothetical sequence of effects, responses, or metabolic events in plants influenced by
`boron was suggested. This is quite an important model as it demonstrates the difficulty
`in determining which steps are effected as a primary and direct result of boron
`interaction and, which are secondary or as a subsequent result of any primary effects.
`
`Other work has been carried out in this area of carbohydrate metabolism, and it has been
`postulated that boron plays a role in plants in the production and translocation of
`sucrose (Dugger & Humphrey, 1960; Loughman, 1961), perhaps by the inhibition of
`starch synthesis (Dugger et al., 1957; Scott, 1960; Augsten & Eichhorn, 1976).
`
`Following the discovery that boron was essential for the growth of vascular plants in the
`1920's boron deficiency was soon identified as being the cause of serious crop losses in
`sugar beet in Germany and apples in both Canada and New Zealand. Since then boron
`deficiency has been recognized in nearly every country and in many crops and the
`addition of boron as a fertilizer along with the 6 other micronutrients, 3 secondary and 3
`major nutrients is well established and it is believed that boron deficiency is more
`widespread than deficiency of any other of the micronutrients. This probably reflects
`the water solubility of borates.
`In addition to any low original amounts of boron,
`borates are depleted by rainfall which washes boron from the topsoil and by cropping
`where it is removed in the harvested material. Natural level in soils can also be
`rendered less available to plants by high levels of calcium and drought. An estimate of
`the global coverage of crops susceptible to boron deficiency can be given as over 205
`million hectares ( compiled from Annon, 1990), although currently the global borate
`micronutrient application is approximately 60,000 product tonnes per annum, mostly in
`the form of pentahydrate borax and disodium octaborate tetrahydrate. This figure has
`grown at a rate of approximately 5 % per year over the last 20 years and is likely to
`continue at the same rate for at least another 20 (Phillips pers. comms).
`
`Probably as a result of the complex biochemical interactions of boron in plants, the
`physiological effects are many fold. Boron is needed for many of the normal functions
`of plant growth, but is particularly involved in meristem development; pollination;
`tissue viability/stability; frost resistance and disease resistance. These have been briefly
`reviewed by Shorrocks (1989).
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`When boron is in short supply normal cell division cannot proceed. Eventually shoot
`and root apical meristems die or become moribund. Boron is essential for proper seed
`set and fruit development and problems here often appear as part of the boron deficiency
`symptoms. It has been established that boron is required for pollen tube growth and the
`boron is adsorbed as the tube grows through the stigma tissue and in some cases pollen
`grain germination is boron dependent.
`
`Apart from the degeneration associated with meristems, breakdown of parenchyma
`tissues are also common in boron deficiency. This is usually manifested by brown
`discoloration, areas or spots of necrosis and the formation of corky nodules and appears
`to be associated with the biochemical effects of overproduction of phenolics and
`lignification.
`
`In a number of situations boron deficiency is also associated with poor frost resistance.
`This is particularly the case in trees such as Pine and Eucalyptus and other studies have
`shown a similar situation with apples and grapes. There is no doubt than boron
`application can significantly increase frost resistance in these situations.
`
`From a microbiological point of view, boron usage in agriculture is particularly
`interesting with regard to disease resistance and control. There have been many
`observations of increased disease resistance following boron fertilizer application,
`including resistance to mildew and aphid attack. It is generally believed that good and
`healthy plant vigour would render the plants less susceptible to attack by pathogenic
`organisms, many of which are highly opportunistic. Other reports have associated the
`enhanced resistance of tomato, capsicum and cabbage to damping off fungi following
`seed treatment with boron, to the increased activities of polyphenol oxidase and
`peroxidase, both of which are influenced by boron (Shorrocks, 1989). Some of the
`observed effects such as the reduction in Ergot caused by the basidiomycete smut
`C/aviceps purpurea on barley and rye (Tainio, 1961; Simojoki, 1969) however, could
`possibly be as a direct fungi static/fungicidal effect and this may also be the case with
`club root in the Brassicaceae caused by Plasmodiophora brassicae (a plasmodial fungus
`in the Gynomyxa).
`
`Boron has been recognized for more than 50 years to be able to influence the impact of
`club root disease. P. brassicae is a soil borne fungal pathogen which exists outside of
`the host as a resting spore which retains viability for many decades. Disease spread is
`by zoospore and the devastating secondary stage of the disease only occurs after
`morphogenesis of the plasmodium into the sporangium containing the zoospores. The
`plasmodium in the host does not appear to cause the plant a problem.
`In a review
`prepared by Dixon (1996) confirmatory evidence obtained in controlled environments
`and in the field shows that the morphogenic change from plasmodium to sporangium in
`the root hair or epidermal cell is inhibited by boron. He concludes however, that it is
`not certain whether this is a direct effect on the fungus or whether it is an effect on the
`host which subsequently effects the fungus life cycle.
`
`An area where borates provide a definite biostatic control of a pathogenic fungus in
`agriculture or rather forestry, is in its use for controlling Fornes disease of conifers.
`This application has been extensively reviewed by Pratt (1996), with recent results in
`the U.K. being summarized by Pratt & Lloyd (1996). Conifers throughout the northern
`hemisphere are susceptible to a root and butt rotting disease caused by the
`
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`Anacor Exhibit 2022
`Flatwing Pharmaceuticals, Inc. v. Anacor Pharmaceuticals, Inc
`IPR2018-00169
`
`
`
`basidiomycete Heterobasidion annosum. The fungus spreads over long distances by
`aerial dispersion of basidiospores, which can be released throughout the year or during
`summer or winter months, depending on ambient temperatures, from fruit bodies on
`rotted wood. In managed forests and plantations, freshly cut stump tops are created at
`regular intervals in thinning and during clear-felling and these are susceptible to
`infection by H annosum basidiospores. The fungus is a primary colonizer of wood, and
`the successful infection of a stump top may lead to almost complete colonization of a
`stump within a few months. This saprophytic phase is of no economic significance per
`se. However, on many soils, the fungus can infect healthy intact roots of standing trees
`where tree and diseased roots are in close contact. Cortical lesions may be so prolific as
`to kill the distal portion of a living root, and thus provide H annosum entry into the
`central xylem of the living tree. From such a position, heartwood in both root and stem
`can be rapidly decayed following colonization by the fungus. This in turn increases the
`risk of premature wind-blow and renders the valuable butt sawlogs useless often to a
`height of many meters. The heartwood decay is cryptic and large numbers of trees can
`be diseased with no outward symptoms until they are felled. Stumps of decayed trees
`are themselves potent sources of infection, since the fungus can remain viable within
`them for many decades (Greig and Pratt 1976). Infection arising from such stumps can
`spread into replacement crops even before thinning provides more stumps as access for
`infection. The disease can therefore increase both within and between rotations.
`
`The disease can, however, be controlled by treatment of stump tops to prevent their
`infection by basidiospores. Because the fungus cannot survive freely in the soil and
`only has access to the stump for a short period of time, the treatment of stumps with a
`prophylactic material is an economically viable and effective strategy against the
`disease. Stump treatment has been used in the U.K. and France to prevent the
`establishment of the disease, in mainly healthy stands, for the past 3