Biological Control of Plant Diseases And Weeds by Kisan Forum Pvt. Ltd.

BIOLOGICAL CONTROL OF PLANT DISEASES AND WEEDS /

PratibhaSharma Division of Plant Pathology Indian Agricultural Research Institute NewDelhi 110012

ICAR

DIRECTORATE OF KNOWLEDGE MANAGEMENT IN AGRICULTURE Indian Council of Agricultural Research Krishi Anusandhan Bhavan I, Pusa, New Delhi 110 012

First Edition

Project Director (DKMA)

Incharge (English Editorial Unit) Editing

Chief Production Officer Assistant Chief Technical Officer

July 2014

Dr Rameshwar Singh Dr ArunaT Kumar Madhu Aggarwal Dr V K Bharti Ashok Shastri

ISBN: 978-81-910388-7-3

Price : ? 600

Published by Dr Rameshwar Singh, Project Director, Directorate of Knowledge Management in Agriculture, Indian Council of Agricultural Research, Krishi Anusandhan Bhavan, Pusa, New Delhi. Lasertypeset at M/s Archana Printographics, 421 -A, Shahpur Jat, New Delhi 110 049 and printed at M/s Chandu Press, D-97, Shakarpur, Delhi-110092

Contents Preface 1.

Introduction

History of biological control Use of microbes as biocontrol agents Biological control of important diseases of major crops Principles and mechanism of biological control Ecology of biological agents 6. Development of bioformulation 7. Successful demonstrations of biological control of 8. important diseases in India Biological control of weeds 9. 10. Guidelines for registration and quality parameters 11. Techniques of molecular typing and gene transfer in biocontrol agents 12. Biocontrol strategies: research and industry applications 13. Biocontrol practicals AppendixI : List of biocontrol agents (BCAs) used against major diseases of important crops Appendix II : Different types of culture media Appendix III : Commercial biocontrol products used for plant disease management worldwide Glossary Index 2. 3. 4. 5.

5 29 72 116 166 176 196 207 220 229 238 248 291

298 302 309 322

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Introduction

Biological controlis defined as the reduction of inoculum density or diseaseproducing activities of a pathogen or parasite in its active or dormant state by one or more organisms, except man, accomplished naturally or through manipulation of environment, host or antagonist or by mass introduction of one or more antagonists. The biological control approach makes use of the invasive plant’s naturally occurring enemies, to help reduce the invasive plant’s impact on agriculture and the environment. Biological control is the deliberate use of one organism to regulate the population size of a pest organism. The term biological control was coined by the late Harry Smith of the University of California in 1919, who defined it as ‘the suppression of insect population by the actions of their native or introduced enemies’. Baker and Cook (1974) described it as ‘the reduction of inoculum density or disease producing activities of a pathogen or a parasite in its active or dormant state, by one or more organisms’. Although there is strong evidence for natural occurrence of biological control on the phylloplane and in the rhizosphere, there are not many examples of field application of themethod. It has been more amenable to its natural occurrence than to mass introduction or manipulation of antagonists except where such plant organs as seed, fruits and seedling’s roots havebeen treated with known antagonists. The term ‘biological control’ commonly refers to the decreased inoculum or the disease-producing activity of a pathogen accomplished through one or more organisms, including host plant (Baker, 1987). The multiplication of pathogen may be suppressed through inhibitory effect by releasing substance that prohibit the growth of the pathogen. In Plant Pathology the term applies to the use of microbial antagonists to suppress diseases as well as the use of the host-specific pathogens to control weed populations. In both fields, the organisms that suppress the pest or pathogen are referred as biological control agent. The term biological controlhas also been applied to the use of natural products extracted or fermented from various sources. These formulations may be very simple mixture of natural

Biological Control of Plant Diseases and Weeds ingredients with specific activities or complex mixture with multiple effects on the host as well as the target pest or pathogen, while such inputs may mimic the activities of the living organisms, non-living inputs should more properly be referred to as biopesticides or biofertilizers depending on the primary benefit provided to the host plant. An important aspect of biocontrol strategies is impact of epidemiological factors on various biocontrol agents in the field. The environment, effectiveness of antagonists and its ability in an ecosystem, host exudates and chemicals are important factors to decide the fate of biocontrol of plant pathogens. Rhizosphere is preferred by the biocontrol agents over phyllosphere for their survival. Biological control is a result of many different types of interaction among microorganisms. Scientists have concentrated on characterization of mechanism occurring in different experimental situations. In all cases, pathogens are antagonized by the presence and activities of the other microorganism. The biocontrol agents antagonize by different mechanism of actions like hyperparasitism, antibiosis, metabolite production, competition etc. The microorganism used in biological control of plant disease is termed as antagonist. An â&#x20AC;&#x2DC;antagonistâ&#x20AC;&#x2122; is a microorganism that adversely affects another growing in association with it. Antagonism is the balance wheel of nature. It operates through competition, parasitism and antibiosis. Biological control is a natural phenomenon, natureâ&#x20AC;&#x2122;s own way of keeping diseases from getting catastrophic. Antagonism includes antibiosis, competition and mycoparasitism, induced resistance and also growth promotion, making it more adaptable. In hyperparasitism, the pathogen is directly attacked by specific biocontrol agent (bca) that kills it or its propagules. Four major groups of hyperparasites have generally been identified which include hypoviruses, facultative parasites, obligate bacterial pathogens and predators. Many microbes produce and secrete one or more compounds with antibiotic activity. Antibiotics are microbial toxins that can, at low concentrations, poison or kill other microorganisms. It has been shown that some antibiotics produced by microorganisms are particularly effective against plant pathogens and diseases caused by them. Many biocontrol active microorganisms produce metabolites that can interfere with growth and activities of pathogen. Lytic enzymes are among these metabolites that can break polymeric compounds, including chitin, proteins, cellulose, hemicelluloses and DNA. Studies have shown that some of these metabolites can sometimes directly result in the suppression of plant pathogen. The nutrient sources in the soil and rhizosphere are frequently not sufficient for microorganisms. For a successful colonization of phyllosphere and rhizosphere a microbe must effectively compete for the available nutrients. On plant surfaces host supplied nutrients include exudates, leachates or senesced tissue. Competition for rare but essential micronutrient such as iron has also been shown to be important in biological control. Iron is extremely limited in the rhizosphere, which leads to competetion.

Introduction

Plant Pathologists have begun to characterize the determinants and pathways of induced resistance stimulated by biological control agents and other nonpathogenic microorganism. The first pathway called systemic acquired resistance (SAR) is mediated by salicylic acid (SA) a chemical compound which is usually produced after pathogen infection and typically leads to the expression of pathogenesis related (PR) proteins. These PR proteins include a variety of enzymes some of which may act directly to lyse invading cells, reinforce cell-wall boundaries to resist infections or induce localized cell death. Second pathway called induced systemic resistance (ISR) is mediated by jasmonic acid (JA) and ethylene, which are produced following applications of some non-pathogenic rhizobacteria. Plant-growth-promoting bacteria (PGPB) triggered ISR fortifies plant cell-wall strength and alters host physiology and metabolic responses, leading to an enhanced synthesis of plant defense chemicals upon challenge by pathogens and/ or abiotic stress factors. Biological control in plant pathology has developed from the knowledge of disease suppression, organic amendments, conservation and introduction of antagonists. The biological pest management includes crop rotation, agronomical operations, plant breeding techniques etc. The use of deliberate introduction of antagonist microflora into the environment to control plant pathogen dominates the present direction in biological control. The biocontrol agents are the entities very well fit in both IPM system and organic farming to reduce pesticide load in the environment and also to produce pesticide free produce which is generally preferred by the consumers. As such biological controlhas become highly relevant for the disease management in crops. Biological weed control is an approach utilizing living organisms to control or reduce the population of an undesirable weed species. Biological control agents can be specific to the weed and cause neither residue problem nor accumulation of toxic products in soil or underground water. Most programmes for the biological control of weeds have been carried out by entomologists who have transported phytophagous insects from native region of the weed. Biological weed control involves the release of organisms and or insect(s) that attack plants to destroy weeds. The aim of biological control is to shift the balance of competition between the weed and the crop in favour of the crop and against the weed. The biological control agent, normally a fungus or insect, may not necessarily kill the target weed but should, at the least, reduce its vigour and competitive ability. From a practical point of view the organism or agent should prevent the weed setting seed or producing other reproductive parts. There is considerable potential for encouraging the use of native biological control agents against weeds. Danielson et al. (1973a) were the first to introduce the concept of mycoherbicide. Since 1982, research efforts towards the development of mycoherbicides have increased tremendously (Charudattan and Dev Vey, 1972; Charudattan, 1991). Most of the effective biocontrol (Fig. 1) strains induce growth of various crops. These responses may be due to suppression of deleterious root microflora, production of growth-stimulating factors, increased nutrient uptake

Biological Control of Plant Diseases and Weeds

Biocontrol Agents

Insect Pathogen

Natural Enemies

Parasitoids

Predators

•

Fungi Bacteria Virus Protozoan

• •

Antagonists

• •

Fungi Bacteria Virus

Fig. 1. Types of biocontrol agents

and enhanced root development helpful in tolerating biotic and abiotic stresses by the plants.

References Baker, A.W. 1987. Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp mediated plant growth promotion. Soil Biology and Biochemistry. 19 : 451.

Baker, K.F. and Cook, R.J. 1974. Biological Control of Plant Pathogen, p 110. University of California Press, Berkeley. Charudattan, R. 1991 . The mycoherbicide approach with plant pathogens, Microbial Control of Weeds, pp. 24-67. D.O. TeBeest (Ed.), Chapman Hall, New York. Charudattan, R. and De Vey, J.E. 1972. Common antigens among varieties of Gossypium hisutum and isolates of Fusarium and Verticillium. Phytopathology. 62 : 230.

Danielson, R.M. and Davey, C.B. 1973a. Nonnutritional factors affecting the growth of Trichoderma in culture. Soil Biology and Soil Biochemistry. 5 : 495-504.

History of Biological Control T Biological control of the plant pathogens, insects and weeds naturally occurs at some level in all agricultural ecosystems, sometimes to a degree where debilitating effects are noticeably reduced. Research interest in dissecting the

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complexity of the interactions that take place in naturally occurring biological control systems has increased in recent years. Biological control is a safe and ecofriendly approach with early history in 200-1887AD. The concept of biocontrol initiated with the insects which was later initiated in intermediate history with the use of microorganisms in form of fungi and bacteria. The beginning of biological control started with the entomologists and commonly understood as standard of biological control. Use of antagonists in field started with introduction of antagonists against insects, than at quite a later stage by microbes against microbes. Last century witnessed biocontrol of plant pathogens by microorganisms. The history of biological control could be divided into two periods i.e. traditional and modem period.

Traditional Period (200

to 1955)

Use of the traditional efforts when living agents were released haphazardly with no scientific approach. Little precise information exists on successes during this time. Some reports of introduction of different microbes are also available roughly from 200 AD to 1887 AD. The traditional period of more discriminating biological control (BC) started with the introduction of the Vedalia beetle, Rodolia cardinalis Mulsant, for control of the cottony cushion scale in 1888; period extended from 1888 to 1955.

200 AD to 1200 AD : (Biological control agents used in augmentation) Chinese were the first to use natural enemies to control insect pests. Nests of the ant Oecophylla smaragdina were sold near Canton in the 3rd century for

Biological Control of Plant Diseases and Weeds use in control of citrus insect pests such as Tesseratoma papillosa (Lepidoptera). Ants were used in 1200 AD for control of date palm pests in Yemen (south of Saudia Arabia). Nests were moved from surrounding hills and placed in trees. Usefulness of ladybird beetles was recognized there in control of aphids and scales

in 1200 A.D.

1300 AD to 1799 AD: (Biological Control was just beginning to be recognized) 1602 AD, Aldrovandi noted the cocoons of Apanteles glomeratus on a parasitized Pieris rapae but thought that cocoons were the insect eggs. In 1706 AD, Vallisnieri interpreted the phenomenon of insect parasitism (parasitoid), however, the honour of being first to understand insect parasitism may belong to microbiologist, Van Leeuwenhoek, who illustrated and discussed a parasite of a sawfly that feeds on willow in a publication is in 1701. The first insect pathogen was recognized by de Reaumur in 1726. It was a Cordyceps fungus on a noctuid. In 1734, de Reaumur suggested to collect the eggs of an ‘aphidivorous fly’ (actually a lacewing) and placed them in greenhouse to control aphids. In 1762, mynah bird, Acridotheres tristis, was successfully introduced from India to Mauritius (off coast of Madagascar) for control of the red locust, Nomadacris septemfasciata. In the late 1700’s, birds were transported internationally for insect control. In 1776, control of the bedbug, Cimex lectularius, was successfully accomplished by releases of the predatory pentatomid Picromerus bidens in Europe. Persoon (1794) first proposed genus Trichoderma and described it. The four species proposed by Persoon were named T. viride, T. nigrescens, T. aureum and T. roseum and were distinguished from each other by their different coloured conidiation. However, these four species are now considered to be unrelated to each other and are presently known as Trichoderma viride, Xylohypha nigrescens (Pen. ex. Fr.) Mason, Sporotrichum aureum Pers ex Fr and Trichothecium roseum (Pers.) Link ex S.F. Gray respectively. The name Trichoderma is now applied to the most frequently encountered green forms typified by the original T. viride species as described by Persoon (1794).

1800 AD to 1849 AD: (During this period advances were made in Europe which were both applied and basic) In 1800, Darwin discussed Tchneumonids’ as natural control factors for cabbage caterpillars. Malthus (England) published Essays on the Principles of Population in 1803. Hartig (Germany) suggested the rearing of parasites from parasitized caterpillars for mass release. Report of Trichoderma viride the conidial stage of Hypocrea rufa, was identified in 1827. The concept of natural control was put forth by Kollar (Austria) in 1837. In 1838, Verhulst described the logistic growth equation but the idea was dormant until 1920 when rediscovered by Pearl and suggested idea of ‘environmental resistance’. The genus Gliocladium was described by Corda inl840. Predators were used for control of the gypsy moth and garden pests in Italy.

History of Biological Control

1850 to 1887: (During this time the focus on biological control switched to the United States) From 1850 to 1870, different types of crops were grown in the United States (especially California) which was initially free from pests. Later farmers saw their crops destroyedby hordes of alien pests. In 1856, AsaFitch (New York) suggested import of parasites from Europe to control the wheat midge, Contarinia tritici. In 1860, parasites were requested from Europe, but none were received. During this period, Benjamin Walsh (Illinois) actively worked for the importation of natural enemies to control the exotic insects in the United States but was unsuccessful. Fortunately, he influenced Charles V. Riley greatly who was in Missouri during Walshâ&#x20AC;&#x2122;s campaign. In 1863, first practical attempt of Biological Control of weeds occurred in India when segments of the prickly pear cactus, Opuntia vulgaris, infested with the imported cochineal insect, Dactylopius ceylonicus, were transported from northern to southernIndia. In 1868, cottony cushion scale, Icerya purchasi Maskell, was introduced into California in around the Menlo Park (CA) area (near San Francisco). Riley conducted the first successful movement of parasites for biological control when parasites were moved from Krikwood, Missouri, to other parts of the state for control of the weevil Conotrachelus nenuphar. In 1870, LeBaron transported apple branches infested with oyster-shell scale parasitized by Aphytismytilaspidis fromGalena to Geneva, Illinois. In 1873, Riley sent the predatory mite Tyroglyphus phylloxerae to France to control the grape phylloxera. The mite was established but did not exert control as expected. In 1882, Trichogramma spp (egg parasites) was shipped from the U.S. to Canada for control of lepidopterous pests. In 1883, the United States Department of Agriculture (USDA) importedApanteles glomeratus from England for control of Pieris rapae (the imported cabbageworm). Parasites were distributed in DC, Iowa, Nebraska and Missouri. This was first intercontinental shipment of parasites. 1888 to 1889: (The Cottony Cushion Scale Project) In 1887, it spread to southern California and was threatening to destroy the infant citrus industry. In 1887, C. V. Riley, Chief of the Division of Entomology, USDA employed Albert Koebele and D. W. Coquillett in research on control of the cottony cushion scale.In 1888, Koebele was sent to Australia to collect natural enemies of the scale. He sent California 12,000 individuals of Cryptochaetum iceryae and 129 individuals of Rodolia cardinalis (the vedalia beetle). Within the year, the cottony cushion scale ceased to be a substantial pest. The vedalia beetle controlled the scale mainly in the inland desert areas and C. iceryae in the coastal areas of California. 1890 to 1899: (Efforts for biological control) In 1912, Koebele went on foreign exploration, but on his return he was recalled from California. Koebele resigned his position and went to work for the Republic of Hawaii until when he retired due to ill health. Due to the success of the Vedalia beetle, great emphasis was placed on importation of coccinelids for

Biological Control of Plant Diseases and Weeds biological control initially in California and Hawaii. It is believed that California and Hawaii got set back many years by promoting mostly biological control projects and not researching alternative control methodologies. In 1894, L.O. Howard replaced C.V. Riley as Chief of the Division of Entomology, USDA. Howard was prejudiced against biological control due to the problems he saw in California. Then, George Compere began as a foreign explorer for California.

1900 to 1930: (New faces and more biological control projects) During 1905-1911, The Gypsy Moth Project started in New England. W. F. Fiske was in-charge in Massachusetts. Howard conducted foreign exploration in Europe and arranged for parasites to be imported to the U.S. Many prominent entomologists were employed on the project, namely Harry Scott Smith, W.R. Thompson, P.H. Timberlake. The Lantana Weed Project was started in Hawaii and is the just first published work on biological control of weeds. Koebele went to Mexico and Central America looking for phytophagous insects which were sent to R.C.L. Perkins in Hawaii. During 1904-20, The Sugarcane Leafhopper Project was also initiated in Hawaii. Hawaiian Sugar Planters Associations (HSPA) created a Division of Entomology in 1904. R.C.L. Perkins was appointed superintendent. Staff consisted of O.H. Swezey, G.W. Kirkaldy, F.W. Terry, Alexander Craw, and Albert Koebele. Later Frederick Muir was employed due to Koebeleâ&#x20AC;&#x2122;s health problems. Muir found the highly effective predator Tytthus (Cyrtorhinus) mundulus (Miridae) in Queensland, Australia, in 1920. Berliner described Bacillus thuringiensis as a causative agent of bacterial disease of the Mediterranean flourmoth in 1911. Prof. H.S. Smith 1913 was appointed superintendent of California State Insectary, Sacramento, CA, in 1913. Facility moved to the University of Californiaâ&#x20AC;&#x2122;s Citrus Experiment Station in 1923 (now UC Riverside). Smith started another facility at Albany, CA, Riverside and Albany (UC Berkeley) made up Department of Biological Control, Univeristy of California (UC) in 1919. United States Department of Agriculture (USDA) laboratory for biological control was established in France. In 1928, The Imperial Bureau of Entomology created the Farnham House Laboratory for biological control work in England in 1927; this was later directed by W. R. Thompson. Historically, biological control of plant diseases has evolved as a natural means of pest management. It has more evolved with insects with gradual development in plant disease management also. As early as 1865 and 1870, de Bary described and named Piptocephalis freseniana and Cicinnobolus cesati and recognized them as mycoparasites (De Bary, 1887). These early papers were soon reported by the report of Brefeld (1872), who described the haustoria and stages in the life history of P. freseniana. Other early studies of parasitic Mucorales were made by VanTieghem (1875) andBainier (1883, 1906). In the United States, Thaxter (1895) was one of the early investigators of Dispira cornuta. Trichothecium roseum Link was first recognized as a facultative parasite as biocontrol agent. There is also a report made by Oudemans and Koning in 1902

History of Biological Control

on Trichoderma koningii, though genus Trichoderma was proposed by Persoon in late 18th Centuary. Some years later Burgeff described the mode of parasitism of Chaectocladium jonesii (Berk, and Br.) Fres and Parasitella simplex Bain on Mucorales. The destructive effects of several fungi on Rhizoctonia solani Kuhn and other soil fungi were reported by other workers (Weindling, 1932; Warren, 1948; Boosalis, 1956). Sanford and Broadfoot were first to use ‘Biological Control’ in plant pathology with 40 bacteria, 24 fungi, organisms or culture filtrates in sterile soil and found organisms more effective against Gaeumannomyces graminis var. tritici. Most of the antibiotics discovered since Fleming’s discovery of penicillin are from soil saprophytic microorganisms. Meanwhile work was initiated against Pythium debaryanum - pine seedling damping-off (Hartley, 1921), Streptomyces scabies - potato scab, grass clippings reduced disease (Millard and Taylor, 1927). The potential of Trichoderma species as biocontrol agents of plant diseases was first recognized in the early 1930s. In 1934, Weindling reported that a strain of T. lignorum produced a ‘lethal principle’ that was excreted into the surrounding medium, allowing parasitic activity by the biocontrol agent. Introduction of antagonistic microorganisms started as early as in the 1930s (Weindling 1932, 1934, Weindling and Fawcett 1936, Weindling and Emerson 1936), it took a backseat for the next several decades.World War II caused a sharp drop in biological control activity.Biological control did not regainpopularity after World War II due to the production of relatively inexpensive synthetic organic insecticides. Entomological research switchedpredominantly to pesticide research. In 1947, the Commonwealth Bureau of Biological Control (CIBC) was initiated, and its headquarters are currently in Trinidad, West Indies. In 1955, the commission Internationale de Lutte Biologique contre les Enemis des Cultures (CILB) was established. This is a worldwide organizaton with headquarters in Zurich, Switzerland. In 1962, the CILB changed its name to the OrganisationInternationale de Lutte Biologique contre les Animaux et les Plants Nuisibles. This organization, also known as the International Organization for Biological Control (IOBC), initiated the publication of the journal ‘Entomophaga’ in 1956, devoted to biological control of arthropod pests and weed species.

The Modern Period: 1956-57 to present The modem period is characterized by more careful planning and more precise evaluation of natural enemies from 1956-57 onward. In 1959, Verm Stern conceived the idea of economic injury level and economic threshold which would permit growers to make informed decisions on when they needed to apply control tactics in their cropping systems and therefore,eliminated the need for scheduled pesticide treatments. In 1962, interest developed nationwide in ecology and the environment after the publication of Rachel Carson’s book Silent Spring. In 1960, this publication helped stimulate

Biological Control of Plant Diseases and Weeds the implementation of the concept of Integrated Pest Management (IPM), and biological control was seen as a core component of IPM. More emphasis was placed on conservation of biological control than classical biological control. In 1964, Paul DeBach and Evert I. Schliner (Division of Biological Control, University of California, Riverside) brought out a publication ‘Biological Control ofInsectPest and Weeds’ which became a major reference source for the biological control community. In 1970, in some areas in the USA (California, North Carolina, Kansas, Texas), IPM scouting was commercializedand natural enemies were relied upon to suppress pests in crops such as cotton, alfalfa, citrus, soybeans, and other crops. In 1970 and 1980, Brian Croft and Marjorie Hoy made impacts by using pesticide resistant natural enemies in cropping systems. In 1983, Francis G Howarth in his landmark paper ‘Biological Control: Panacea or Pandora’s Box’ significantly impacted classical biological control efforts by concluding that classical biological control of arthropods significantly contributed to extinction of desirable species (e.g. endemic). Howell and Stipanovic in 1983 isolated and described a new antibiotic, gliovirin, from Gliocladium (Trichoderma) virens (GVP) that was strongly inhibitory to Pythium ultimum and Phytophthora species. The fungus Trichoderma inhamatum was described by Veerkamp and Gams in 1983. Research on biocontrol of plant pathogens received attention in 1963 in an international symposium held at the University of California, Berkley, where the publication entitled Ecology of soil borne plant pathogens-Prelude to biological control was presented. In this symposium, Rishbeth presented a paper on biological control of Annonus root rot of pine with Phlebia gigantea applied to freshly cut stumps to preempt colonization of the stumps by the pathogen Heterobasidium annosum. This was followed by the development of Agrobacterium radiobacter K84 by Kerr in 1980 which further directed the use of antagonists. Gerlagh (1968) worked for four successive crops in soil, increased suppression of Gaeumannomyces graminis var. tritici and also found that steaming destroys antagonists. R. James Cook, David Weller, Linda Thomashow from USDA/ARS, WSU found that 2fluoroglucinol from Pseudomonas fluorescens, and P. aureofaciens have an important role in biocontrol. In the 1990’s, two additional biological control journals appeared, Biological Control- Theory and Application in Pest Management (Academic Press) and Biological Science and Technology (Carfax Publishing). Entomophaga changed its name to Biocontrol in 1997.

TAXONOMY OF IMPORTANT BIOCONTROL AGENTS Trichoderma In 1969, Rifai provided the first real generic description of Trichoderma, based on colony growth rate and microscopic characters. Rifai divided the genus into nine species, distinguished from each other primarily by conidiophore branching patterns and conidium morphology. However, Rifai recognized that the groupings that he defined were made up of more than one genetic entity.

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