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Identification of Pathogenic Fungi from Soybean

Luca Riccioni1, Kristina Petrovi2 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per la Patologia Vegetale, Via C. G. Bertero 22, Rome, Italy 2 Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia

Introduction
A large number of microorganisms are parasites of soybean (Glycine max (L.) Merr.), and they cause various pathological changes in all organs of the plant. Soybean diseases can significantly affect yield level, the quality and the stability of this industrial crop. In addition, epiphytotic attacks by some pathogens may bring into question the profitability of soybean growing. More than 135 microorganisms have been described on soybean, but only about 30 species belong to the group of economically important pathogens (Roy et al., 2000). In some agroecological regions several parasites appear in high intensities on soybean, while in others these are either not present or occur sporadically. Which will be the dominant parasite in a particular region depends primarily on climate factors. It has been found that the most important fungal parasites in Serbia are Peronospora manshurica (downy mildew) on leaves; Diaporthe phaseolorum var. caulivora (northern stem canker) and Sclerotinia sclerotiorum (white mold) on stems; Macrophomina phaseolina (charcoal rot) on root, and Diaporthe/Phomopsis species which are the main causes of seed decay. In addition to these, there are also many other pathogens, occurring sporadically and rarely cause significant damage (Vidi et al., 2008). It should be noted that in some regions of the world very important soybean diseases occur, such as Phytophthora root and stem rot (Phytophthora sojae), soybean rust (Phakopsora pachyrhizi), sudden death syndrome (Fusarium virguliforme), southern stem canker (D. phaseolorum var. meridionalis) and powdery mildew (Microsphaera diffusa), but the presence of these parasites has not been observed on soybean in Serbia. Accurate diagnosis is essential in understanding plant diseases which is a precondition for taking effective control measures. A susceptible host plant, a pathogen and a favourable environment are the three factors composing the plant disease triangle. Only in the common interaction these factors cause disease. Diagnosis of plant diseases based only on the symptomatology is often unreliable. First of all, it is necessary to determine whether it is an infectious disease (biotic) or a non-infectious disease (abiotic), based on visible reaction or symptoms. In some cases, when the typical symptoms are visible and followed by the presence of pathogens, such as fungi reproductive organs, it is relatively easy to identify which parasite is present. However, many pathogens can cause non-specific and variable symptoms, or latent infection, without visible signs of diseases. For these reasons, in most cases, it is necessary to perform detail study of symptoms, and take one step further in order to establish a correct and reliable diagnosis. Therefore, the diagnosis of the disease involves complete identification of the pathogen, which involves conscientious, comprehensive and detailed research. Progress in the development of knowledge on plant pathology nowadays provides a number of methods by which diseases can be discerned. In addition to conventional methods, the development of molecular biology techniques, which are based on the detection of pathogen genomes, has provided new ways of identification and characterization. Molecular methods, such as nucleic acid hybridization and polymerase chain reaction (PCR), have made a breakthrough in modern diagnostics of plant disease pathogens. This paper will review the common conventional and molecular methods currently used for identification of fungi with special emphasis on soybean pathogens. The paper is divided into two parts; the first lists common methods that are used in the process of detection and identification of pathogens, while the second presents the identification of the most important pathogens of soybean. 342

Proceedings of plant pathogens detection and diagnosis of diseases


The first step in diagnosing a disease is to determine whether the disease is of infectious or noninfectious nature. If the disease is caused by biotic factors (infectious nature), it is necessary to examine the connection of the pathogens with the diseased plant host to be sure that the pathogen agent of diseases is found and not some contaminant (secondary infection or saprophytic). The procedures applied in this case are known as Kochs postulates, which consist of four criteria: (1) the pathogen must be present on all diseased plants, (2) the pathogen must be isolated from a diseased plant and grown in pure culture, (3) the cultured pathogen should cause disease when introduced into a healthy test plant (reproduction of identical symptoms - pathogenicity) and (4) the pathogen must be reisolated from the inoculated and diseased host and identified as being the same inoculated pathogen. The disadvantage of this procedure is that it is based only on the specific symptoms, but the absence of symptoms does not mean the absence of the pathogens, because they may be present in latent form. For this reason, there is an increasing interest in developing new, faster and sensitive method for the identification of the pathogens and diagnosis of diseases (Fig. 1).

Figure 1. Diagnosis of plant diseases

In todays laboratory, the ability to detect and identify pathogens has undergone major changes. The development of molecular methods that rely on the detection of genomic elements (DNA or RNA) with or without culture has led the way in this change. Some of the main reasons for this change from phenotypic to molecular testing include issues as the slow growth of pathogens, the detection of organisms that exhibit biochemical characteristics that do not fit patterns of known species, and the inability to detect non-cultivatable organisms, such as the obligate parasite P. manshurica. Although culture based methods are still considered the gold standard for identification and diagnosis, molecular methods have emerged as the confirmatory method for identification in many diagnostic applications. 343

Identification of Pathogenic Fungi from Soybean

It should be noted that conventional and molecular methods do not have the same importance in the examination of each pathogen. In other words, in the determination of some pathogens conventional methods have increasing importance over molecular and otherwise. So, in the identification of Diaporthe/ Phomopsis species, in addition to conventional, molecular methods are now widely used. The reason for this is primarily the presence of morphological variability within the Diaporthe/Phomopsis complex. Also, identification of Diaporthe/Phomopsis species by conventional methods takes several weeks, even months, because the fungi slowly fructify. On the other hand, in the detection of the pathogen P. manshurica, S. sclerotiorum and M. phaseolina, molecular techniques have no significance and their identification is reduced to conventional methods. For determination of S. sclerotiorum and M. phaseolina only a few days are sufficient. Both pathogens are polyphagous organisms, characterized by very aggressive and fast developing. Also, an important feature of these pathogens is the formation of sclerotia (S. sclerotiorum) and microsclerotia (M. phaseolina), which are the primary key of determination. However, although molecular methods have no great application in the detection of these fungi, they have found application in the study of variability within the pathogen population. Also, application of molecular methods in the detection of P. manshurica could be irrelevant, because the symptoms of the pathogen are very clear and characteristics. However, these methods are particularly important in the study of race composition of the pathogen. There is great variability within the fungal population. Until now more than 34 different physiological races have been established and their number is constantly increasing.

Identification of pathogen
The first step in the identification of fungi is to describe the symptoms of the diseased plants (Table 1). Then choose a method for the isolation and development of fungi in artificial medium to determine the species. Finally, the obtained isolate, which manifest pathogenic characteristics, is identified using morphological, cultural and molecular criteria.
Table 1. List of identified pathogenic fungi on soybean in Serbia Mycosis of seedlings Pythium spp. Pythium root rot Rhizoctonia solani* Rhizoctonia root rot Fusarium spp.* Fusarium wilt and root rot Mycosis of root and lower part of stem Macrophomina phaseolina Charcoal rot Mycosis of stem Diaporthe phaseolorum var. caulivora Stem canker Diaporthe phaseolorum var. sojae Pod and stem blight Sclerotinia sclerotiorum White mold Colletotrichum destructivum, C. truncatum Anthracnose stem blight Phialophora gregata Brown stem rot Mycosis of leaf Peronospora manshurica Downy mildew Cercospora kikuchii Cercospora leaf blight & Purple seed stain Cercospora sojina Frogeye leaf spot Septoria glycines Septoria brown spot Phyllosticta sojaecola Phyllosticta leaf spot Alternaria alternata, A. tenuissima, A. atrans Alternaria leaf spot Corynespora cassiicola Target spot Ascochyta sojaecola Ascochyta leaf and pod spot Mycosis of seed** Phomopsis longicolla Phomopsis seed decay

* May occur in the later growth stages of soybean on pods and stem ** P  . longicolla is the most important parasite of seeds, but seeds can also be colonized by other Diaporthe/Phomopsis species, as well as Fusarium spp., C. kikuchii, Alternaria spp., P. manshurica, S. sclerotiorum and M. phaseolina.

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Morphological characteristics
The identification of fungi is based upon an evaluation of their colony characteristics as well as their microscopic morphology. Visual examination of the colony will reveal important data concerning texture, topography and pigments. A specific terminology is used to describe common colony types, such as: form, size, elevation, margin, surface, opacity and colour (Fig. 2).

Figure 2. Morphology of colony (http://www.sciencebuddies.org)

Structures of mycelium
The thallus of fungi is composed of thin filaments called hyphae. There are three types of hyphae: (1) vegetative hyphae, which absorb the nutrients from the culture medium; (2) aerial hyphae, which grow above the agar and give the colony texture; and (3) fertile hyphae, which produce the reproductive structures. Hyphae may or may not produce cross-walls, called septations. Many hyphae tangled together into a thick mass are called a mycelium. The fungus may also form somatic structures, which enable the fungus to survive a period of stress, such as sclerotia and stromata (Carris et al., 2012). Also, the fungus may escape from reduced resources using aggregating hyphal structures called strands or rhizomorphs. The aggregated hyphae grow beyond the zone of depletion and in doing so, may locate further resources. Each of these structures is constructed from modified hyphae.

Formation of fruiting bodies


Subkingdom Dikarya includes two of the best-known phylums of fungi, the Ascomycota and the Basidiomycota, which differ based on fruiting bodies. The ascomycete fungi form various types of ascocarp: perithecium, pseudoperithecium, cleistothecium, chasmothecuma and apothecium; while basidiomycete fungi form different types of basidiocarp (Carris et al., 2012).

Formation of spores
Spores are one of the most important indicators used to identify phytopathogenic fungi. The fungi form a wide range of different types of spores following asexual or sexual processes. Fungi produce two major types of asexual spore: sporangiospores (endogenous spores) and conidia (exogenous 345

Identification of Pathogenic Fungi from Soybean

spores) (Carris et al., 2012). Sporangiospores are divided into two groups: zoospores (motile) and aplanospores (non-motile), while the conidia have two main types of spore: thallic (arthrospores or oidia and chlamydospores) and blastic (blastospores, porospores, aleuriospores, annellospores and phialospores). On the other hand, some fungi also have sexual sporulation, which involves formation of oospores, zygospores, ascospores or basidiospores.

Selective medium
Some plant pathogenic organisms may be cultured in vitro. For these, placing infected material on a suitable medium and inspecting the resulting colonies of the pathogen may be sufficient for a positive identification. However, the choice of medium is important. Routine mycological diagnostic for culturing of fungi recommends potato dextrose agar (PDA) as the basic medium for the isolation of fungi. In addition, there is the selective medium, and considerable effort has been made for some pathogens or groups of pathogens to adapt substrates so that only the organisms of interest will grow.

Biochemical markers
All organisms have distinctive biochemical features and these can be used for diagnosis. Some characteristics are shared by large groups while, at the opposite end of the scale, others are unique to individual populations. The choice of character is therefore paramount in determining the taxonomic level to which an organism is defined. Biochemical markers, such as fatty acids, proteins, carbohydrates and secondary metabolites, can be used in chemotaxonomy of fungi.

Secondary metabolites
Fungal chemotaxonomy based on secondary metabolites has been used successfully in large ascomycete genera such as Alternaria, Aspergillus, Fusarium, and Penicillium (Frisvad et al., 2008) and has been suggested for use in Colletotrichum (Abang et al. 2009; Cai et al. 2009). Phytotoxins are a class of secondary metabolites (natural products) produced by microbes that have deleterious effects on plants. There are commonly divided into specific and non-specific, but depending on the pathogenetic role, phytotoxins are divided into pathotoxins and vivotoxins (Berestetskiy, 2008). Pathotoxins are prerequisite to plant infection by necrotrophic pathogens, while vivotoxins are synthesized by pathogens in infected plant tissues. A considerable number of phytotoxins are toxic to animals and humans. Depending on the economic importance, they are classified as mycotoxins or antibiotics (Berestetskiy, 2008). Phytotoxic metabolites are produced by fungi of diverse taxa and ecological groups. It is known that S. sclerotiorum and M. phaseolina are capable of producing a number of phytotoxins. Oxalic acid, sclerin, sclerolide, sclerotinin A, sclerotinin B, sclerone and isosclerone are produced by S. sclerotiorum, while asperlin, isoasperlin, phomalactone, phaseolinic acid, phomenon, phaseolinone and (-)-botryodiplodin are produced by M. phaseolina (Sett et al., 2000; Ahiahonu, 2003; Ramezani et al., 2007). Pathogens from Diaporthe/Phomopsis complex belong to the group of endophytes and they are a rich source of secondary metabolites (Tan & Zou, 2001; Firkov et al., 2007, Guo et al., 2008). For example, D. phaseolorum var. sojae forms four types of cytochalasin, while P. longicolla produces three types of dicerandrol (Cole et al., 1982; Wagenaar & Clardy, 2001). Also, it was found that D. phaseolorum var. caulivora, D. phaseolorum var. sojae, and P. longicolla produce one or more metabolites, which are phytotoxic to seed germination and seedling of soybeans (Ivanovi & Sinclair, 1989), while Lalitha et al. (1989) reported that D. phaseolorum var. caulivora produces toxin which causes typical symptoms of stem canker on soybean. However, these metabolites have not been identified.

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Serological method
Serological techniques are important for diagnosing of fungal pathogens especially for laboratories without availability to molecular tests. Assays detect surrogate markers of infection such as fungal antigens and metabolites or antibodies to fungal antigens. One or more of these approaches may provide diagnostic clues as well as an insight into the progression of infection (Lau et al., 2009). Immunological method ELISA (Enzyme Linked Immunosorbent Assay) has successfully been applied in the detection of latent infection of soybean seeds with Diaporthe/Phomopsis species (Gleason et al., 1987; Brill & Sinclair, 1990; Velicheti et al., 1993).

Techniques of nucleic acid


Most studies of phytopathogenic fungal strains and populations focused on phenotypic differences in morphology and physiology, and when possible, mating. However, these phenotypic features are often difficult to observe, quantify, standardize, and/or analyze. In recent years, there has been substantial progress in the development of innovative methods to analyze fungi (and other organisms) at the molecular level. Molecular methods are in partial agreement with phenotypic and genotypic groups and some have been used in infraspecific taxonomy. Here is review of the molecular methods used for the identification and characterization of major soybean pathogens with special emphasis on Diaporthe/Phomopsis complex, because molecular techniques have been applied mostly within this genus.

Random amplified polymorphic DNA (RAPD)


One of the first molecular markers which were applied in the analysis of genetic variation, phylogenetic relatedness, genome mapping and evaluation of diversity, were RAPD markers (Bruns et al., 1991; Ivanovi et al., 2004; Setti et al., 2011). This method represents PCR technique that detects nucleotide sequence polymorphisms in the DNA molecule by using arbiter short PCR primers. RAPD markers were first applied to characterize the species D. phaseolorum and P. longicolla (Fernndez & Hanlin, 1996). Based on the obtained profiles, the isolates were divided into two species, P. longicolla and D. phaseolorum, provided that within the species D. phaseolorum three varieties were identified: D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis and D. phaseolorum var. sojae. It was found that P. longicolla, D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis give a specific profile in contrast to D. phaseolorum var. sojae, which was highly variable and provided many different profiles. Also, it was reported that RAPD markers are suitable for measuring genetic relatedness and detecting variation within and between M. phaseolina populations from different hosts (Fuhlbohm, 1997; Almeida et al., 2003; Jana et al., 2003; Rayatpanah et al., 2012).

PCR-Restriction fragment length polymorphism (PCR-RFLP)


In order to distinguish Diaporthe/Phomopsis species from other fungi that parasitize soybean PCRRFLP technique was successfully applied (Zhang et al., 1997). This method includes amplification of specific DNA and digestion of restriction enzymes (endonucleases). Restriction enzymes cut DNA at specific places and give fragments of different sizes, which give a different profile when separated by gel electrophoresis. Profile combinations of two or more restriction enzymes provide a unique molecular profile for each species, which is a molecular determinant. Using this method molecular identification and phylogenetic grouping of D. phaseolorum and P. longicolla was carried out, which were isolated from plant tissues and seeds of soybean (Zhang et al., 1998; 1999; Riccioni et al., 1998; 2003; 2005). The DNA was amplified by universal primers ITS5 and ITS4, and digestion was carried out with five restriction enzymes (AluI, RsaI, HhaI, MseI and ScrFI). Restriction enzymes showed the 347

Identification of Pathogenic Fungi from Soybean

specific electrophoretic profile for D. phaseolorum var. meridionalis, D. phaseolorum var. caulivora and P. longicolla, while for D. phaseolorum var. sojae various combinations were observed, but always different from combinations of previously mentioned varieties. Morphological identification was carried out in addition to molecular methods, but it was only possible in 80% of the analyzed isolates, because isolates often become sterile after prolonged storage on culture media. The results of these studies suggest that the P. longicolla is individual species; D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora are varieties of D. phaseolorum, while D. phaseolorum var. sojae is either several varieties of D. phaseolorum or possibly several distinct species (Zhang et al., 1998; Riccioni & Vrandei, 2007). We used the universal primers ITS5 and ITS4 for amplification of 600 bp from ITS region to distinguish isolates of P. longicolla, D. phaseolorum var. caulivora, and D. phaseolorum var. sojae from soybean in Serbia (Table 2). Also, we used four isolates of D. phaseolorum var. meridionalis originating from the USA. Digestion of PCR product with four different restriction enzymes (AluI, RsaI, HhaI and MseI) gave unique molecular profiles for each species (Fig. 3). The sequence of the ITS region of several Diaporthe/Phomopsis isolates confirmed the results of the PCR-RFLP method (Fig. 4).

Figure 3.  Electrophoretic profiles (A, B, C) of Diaporthe/Phomopsis species obtained by digestion with AluI, RsaI, HhaI and MseI restriction enzymes.

Figure 4. Electrophoretic profiles (A, B, C) of ITS sequences of Diaporthe/Phomopsis species obtained by virtual digestion with AluI, RsaI, HhaI and MseI restriction enzymes

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Table 2. Isolates used for PCR-RFLP and phylogenetic analyses


Host Origin Collector

Species

Isolate

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Psidium guajava Rio de Janeiro, Brazil

D. phaseolorum: var. caulivora var. meridionalis var. sojae P. longicolla Glycine max G. max G. max G. max G. max G. max G. max G. max Aspalathus linearis G. max G. max G. max G. max Stokesia laevis Helianthus annuus Caperonia palustris G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max G. max A.O. Carvalho JN100601 Ohio, USA Osijek, Croatia Ohio, USA Novi Sad, Serbia Novi Sad, Serbia Novi Sad, Serbia Novi Sad, Serbia Georgia, USA Clanwilliam, South Africa Arkansas, USA Georgia, USA Georgia, USA Georgia, USA Mississippi, USA Ostrovo, Croatia Mississippi, USA Serbia Novi Sad, Serbia Sombor, Serbia Subotica, Serbia abac, Serbia Ohio, USA Sopot, Croatia Georgia, USA Novi Sad, Serbia Novi Sad, Serbia Novi Sad, Serbia K. Kmetz K. Vrandei A. F. Fernndez M. Vidi K. Petrovi K. Petrovi K. Petrovi A. F. Fernndez J.C. J. Van Rensburg J. Rupe A. F. Fernndez A. F. Fernndez A. F. Fernndez F. A. Uecker K. Vrandei A. Mengistu D. Rekab M. Vidi M. Vidi M. Vidi K. Petrovi T. W. Hobbs K. Vrandei A. F. Fernndez K. Petrovi K. Petrovi M. Vidi

ATCC:28464 CBS:127268 Dpc 313 DPC31 DPC/KR1 DPC/KR15 DPC/KR21 ATCC:200236 = Dpm 1F CBS:117169 STC-8 Dpm 201 Dpm 421 Dpm 664 ATCC:64802 = FAU458 CBS:127266 CBS:116019 10-99 PS63 PS69 PS73 PS/KR3 ATCC:60325 = FAU600 CBS:127267 Dps 7 PL/KR19 PL/KR3b PDS157A

GenBank accession numbers ITS TEF1- EF594039 (-) HM347712 HM347691 JQ697851 JQ697864 JF418935 JF461466 JF418936 JQ697852 JF418937 JF461467 JF418934 JF461465 JF430486 JF461480 DQ286275 DQ286249 JF495106 AF398893 JQ697850 JQ697863 JQ697849 JQ697862 JF430485 JF461479 U11323 + U11373 TreeBASE HM347707 HM347672 AY745024 (-) AJ312359 (-) JQ697848 JQ697861 JQ697847 JQ697860 JQ697846 JQ697859 JF430489 JF461471 U11357 + U11411 AF398896 HM347700 HM347685 JQ697844 JQ697857 JF430483 JF461469 JF430482 JF461468 JQ697845 JQ697858 JN099031

Cylindrocladiella peruviana

CBS:116103

ATCC, American Type Culture Collection, Manassas,VA, USA; CBS, The Centraalbureau voor Schimmelcultures, Utrecht, Netherlands. Newly generated sequences are in bold. The TEF1- sequence of isolate ATCC:64802 = FAU458 was not available from GenBank and was retrieved from TreeBASE study S1506 (Janse van Rensburg et al., 2006).

Identification of Pathogenic Fungi from Soybean

Species-specific probes
Oligonucleotide specific primers or probes targeting the ITS region have been demonstrated to selectively detect several agriculturally important fungi. Species specificity is an important criterion for DNA-based diagnosis. Zhang et al. (1997) developed Phomopsis-specific primers Phom I and Phom II from polymorphic regions of DNA for the amplification of 337 bp from the ITS region to distinguish isolates of P. longicolla and D. phaseolorum from other soybean pathogens. None of the amplified products was observed in the DNA of seven other soybean fungal pathogens or soybean plant genomic DNA. These specific primers have two main applications: i) detection of Diaporthe/Phomopsis species during latent infection of soybean plants and seeds, and ii) seed testing in laboratories where molecular detection may be more reliable and faster method than conventional morphological examination of seeds on agar. Based on the known ITS sequences of rDNA three TaqMan sets were also developed (PL-3, PL-5 and DPC-3) for detection of Diaporthe/Phomopsis pathogens from soybean seeds. This method is highly sensitive, reliable and the fastest technique for detection of pathogens based on PCR (Zhang et al., 1999). A rapid PCR-based detection method was developed for identification of P. manshurica directly from total DNA extracts of infected plants. Specific primers PM3 and PM4 were designed based on ITS sequences for amplification of 283 bp and used to distinguish P. manshurica from other soybean pathogens (Lai et al., 2004). However, studies of the molecular variability of P. manshurica populations have not been conducted, which would be very interesting considering that within this fungus identified a large number of physiological races. The specific primers MpKR1 and MpKF1 used for amplification of 350 bp from ITS region of M. phaseolina (Babu et al., 2007). The absence of 350 bp product in other species of soilborne fungi, bacteria and actinomycetes confirmed that the primers can be utilized selectively and specifically to identify M. phaseolina.

Molecular phylogenetic approach


As we have seen, the most common region of DNA used in diagnostic procedures and phylogenetic studies of fungi is nuclear ribosomal DNA (nrDNK). Within this region there are conservative genes that encode 18S, 5.8S and 28S rRNA molecules. Between these three genes there are internal transcribed spacers (ITS1 and ITS2), while two rDNA units are separated by intergenic spacers (IGS). These regions (ITS and IGS) are highly variable and are used for the detection of species and lower taxonomic units (Henson & French, 1993; Setti et al., 2011). In phylogenetic studies, in addition to the ITS region, often used sequences of small (SSU) and large (LSU) ribosomal subunit genes as well as sequences of genes responsible for the synthesis of -tubulin and TEF1- (Translation Elongation Factor 1 alpha) protein. Genes of small (18S) and large (28S) ribosomal subunit as well as TEF1- gene, are highly conserved, which is desirable feature for phylogenetic conclusions (Roger et al., 1999). One or more of those genes are candidates to became in the near future the barcode for the identification of fungal species (www.fungalbarcoding.org). Also, the phylogeny of fungi often is reconstructed by using the mating-type (MAT) locus (Turgeon, 1998). This locus regulates sexual reproduction in fungi and based on this it can be known which species are self-fertile (homothalic), and which are self-sterile (heterothalic). In other words, homothalic species have sexual reproduction, because their genome contains two types of MAT locus. In contrast, heterothalic species have only one type of MAT locus, so for sexual reproduction it is necessary to cross with species that has a compatible MAT locus (Turgeon, 1998). This means that species with same MAT locus will not form diploid cells, and therefore cannot give fertile progeny. In Diaporthe and Phomopsis species, MAT locus differs in only one gene (Santos et al., 2010; 2011). For the purpose of diagnosis of mating-type in these species and evaluate their usefulness in inducing teleomorph in vitro, primers for amplification of MAT1-1-1 and MAT1-2-1 genes were developed. The conducted research has shown that MAT phylogeny is completely matched with phylogeny of TEF1- gene (Santos et al., 2010; 2011). 350

ITS and TEF1- sequences, summarized in Table 2, were used for phylogenetic grouping of Diaporthe/Phomopsis species, which were performed in PAUP * 4.0b10 (Swofford, 2002) by Neighbor Joining (NJ) and Maximum Parsimony (MP) methods. TEF1- sequences were not available for all taxa for which ITS sequences could be downloaded from GenBank. However, the partition homogeneity test indicated phylogenetic congruence between ITS and TEF1- genes for all characters (P = 1.00), and therefore the phylogenetic tree was generated for all of them using combined data matrix. NJ phylogram was reconstructed using the K2P (Kimura two-parameter) substitutional model, while the MP phylogram reconstructed using a heuristic search option with TBR branch swapping and 1,000 random sequence additions. Gaps are treated as missing character in MP. In both phylogenetic methods were performed bootstrap analyses of 1000 replications (Felsenstein, 1985). The matrix contains 28 taxa, including Cylindrocladiella peruviana CBS:116103 as outgroup. Sequences are listed by their isolate number in the tree, while newly generated sequences are in bold. The NJ analysis resulted in a tree with the same topology as the MP analysis (Fig. 5). MP bootstrap values are shown above the branches and NJ bootstrap values are shown below the branches. Phylogenetic tree indicates that the analyzed Diaporthe/Phomopsis isolates divided into four clades with bootstrap support values > 99% for MP and > 94% for NJ. It can be concluded that a complex of soybean diseases is caused by P. longicolla and three varieties of D. phaseolorum. Since the phylogenetic tree clearly distinguishes each species, which coincides with the results of PCR-RFLP method, it can be concluded that PCR-RFLP method is a quick and reliable identification method for Diaporthe/Phomopsis species from soybean.

Figure 5. R  econstructed phylogenetic tree based on combined data matrix of ITS and TEF1- sequences. Tree length (TL) = 618; Consistency index (CI) = 0.9337; Homoplasy index (HI) = 0.0663; Retention index (RI) = 0.9767; Rescaled consistency index (RC) = 0.9119.

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Identification of Pathogenic Fungi from Soybean

Identification of Peronospora manshurica


P. manshurica is an obligate fungus that causes downy mildew disease. It is the host specific pathogen, which will not attack other plant species except soybeans. Systematic position. Domain Eukaryota, supergroup Chromalveolata, kingdom Heterokontae, phylum Oomycota, class Oomycetes, order Peronosporales, family Peronosporaceae, genus Peronospora. Symptoms. The initial symptoms of downy mildew caused by P. manshurica are small pale green or yellowish spots, which necrose and merge with time (Fig. 6). On the reverse side of leaf tan to gray cover form of conidiophores and conidia can occur, especially under wet and humid conditions. Pods may be infected without any symptoms, and seeds are partly or completely encrusted with white mycelia and oospores (Fig. 6).

Figure 6. Symptoms of downy mildew on leaf and seeds

Morphology. P. manshurica is biotrophic obligate pseudofungus. Oospores are the survival propagules and initiate primary infections (Fig. 7). Oospores result from the fusion of oogonia and antheridia. They germinate and gives initial hypha, which is unicellular (non septate). The mycelium develops in the intercellular space of the host plant, penetrating the cells by cylindrical, curved and branched haustoria. Conidiophores branch dichotomously and at the end are two sterigmata, each carrying one elliptical or oval conidium (Fig. 7). Conidia germinate in drop of water and initiate secondary infections.

Figure 7. P. manshurica. A - Oospores; B - Dichotomous branching (www.apsnet.org)

Physiological specialization. High variation in pathogenicity has been observed within the population of P. manshurica. More than 34 different physiological races have been identified based on the reaction of several soybean cultivars (Marcinkowska, 1991). Characteristic of this fungus is that the number of physiological races is constantly increasing. 352

Identification of Sclerotinia sclerotiorum


S. sclerotiorum is an economically important necrotrophic fungal pathogen with a worldwide distribution of more than 400 dicotyledonous host species. Included among these hosts are several agriculturally important plants, including soybean, sunflower and oilseed rape. Systematic position. Domain Eukaryota, kingdom Fungi, subkingdom Dikarya, phylum Ascomycota, subphylum Pezizomycotina, class Leotiomycetes, order Helotiales, family Sclerotiniaceae, genus Sclerotinia. Symptoms. Initial lesions of white mold disease caused by S. sclerotiorum usually develop at stem nodes during or after flowering. The infected stem tissue becomes soft and watery, and the whole plant rots in moist conditions. The infected plant parts become covered with cottony mycelium and black sclerotia develop on or inside infected stems and pods (Fig. 8).

Figure 8. Symptom of white mold

Morphology. S. sclerotiorum is a necrotrophic homothallic pathogen. The fungus produces white aerial mycelium, which is hyaline, branched and multinucleate consisting of closely septate hyphae. No asexual conidia are produced; however, microconidia are produced on hyphae or the apothecial hymenium (Kohn, 1979). Nevertheless, these microconidia do not germinate and their role in the biology of the fungus is still unknown. The black sclerotia forms from the aggregation of the mycelial hyphae and they play a major role in disease cycles (Fig. 9). Sclerotia are the primary survival structure and are capable of remaining viable for several years in soil. They can germinate carpogenically or myceliogenically, depending on environmental conditions, resulting in two distinct categories of diseases (Bolton et al., 2006). Sclerotia that germinate myceliogenically produce hyphae that can directly attack plant tissues (Bardin and Huang, 2001), while sclerotia that germinate carpogenically produce apothecia and subsequently ascospores that infect aboveground portions of host plants.

Figure 9. S. sclerotiorum. A - colony and sclerotia on PDA; B - sclerotia; C - apothecia (www.bulletin.ipm. illinois.edu).

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Physiological specialization. A lack of variation in virulence among isolates from defined geographical areas has been reported in a number of studies on agricultural populations (Atallah et al., 2004; Auclair et al., 2004; Kull et al., 2004; Sexton and Howlett, 2004). Differences in virulence may be detected when comparing isolates from widely separate geographical regions. There has been no conclusive evidence to suggest host specialization among isolates of S. sclerotiorum (Kull et al., 2004). However, comparison of S. sclerotiorum populations on cultivated oilseed rape and on the wild perennial host Ranunculus ficaria indicated major differences between agricultural and wild populations (Kohn, 1995). For example, DNA fingerprint diversity was high in agricultural populations but low in wild populations and there was no evidence of outcrossing in agricultural populations even though recombination occurred in wild populations.

Identification of Macrophomina phaseolina


M. phaseolina is a necrotrophic soilborne fungal pathogen that causes a disease commonly known as charcoal rot. This fungus has the potential to infect over 500 different plant species worldwide including many important crops such as soybean, corn and sorghum. Systematic position. Domain Eukaryota, kingdom Fungi, subkingdom Dikarya, phylum Ascomycota, subphylum Pezizomycotina, class Dothideomycetes, order Botryosphaeriales, family Botryosphaeriaceae, genus Macrophomina. Symptoms. The typical symptoms of disease appear during or after flowering. The infection first occurs on plant roots as light brown spots, which latter spread to the entire root system, lower part of stem, lateral branches, and in favourable conditions covers a larger part of the plant. Surfaces of the root and stem become light grey (silvery discoloration), while numerous black microsclerotia develop beneath the epidermis, which give them a charcoal-sprinkled appearance (Fig. 10).

Figure 10. Symptom of charcoal rot

Morphology. M. phaseolina is a soilborne plant pathogen belonging to the group of extremely thermophilous fungi. It forms a sparse whitish mycelium, which is septate and branched, and black microsclerotia (Fig. 11). Microsclerotia germinate in the soil, each producing several initial hyphae. When they come in the contact with soybean roots, the initial hyphae form appressoria with infection 354

hyphae that penetrate the root. In addition to microsclerotia, the pathogen also forms pycnidia with conidia, but pycnidial stage is not common on soybean.

Figure 11. M. phaseolina. A - colony on PDA; B - microsclerotia (www.padil.gov.au).

Physiological specialization. The genetic diversity of M. phaseolina could favour its survival and adaptation to variable environments, because significant morphological, physiological, pathogenic, and genetic diversity has been reported (Rayatpanah et al., 2012). However, no clear evidence to suggest formae specials, subspecies or physiological races has been reported, although Su et al. (2001) suggested host specialization in the genus.

Identification of Diaporthe/Phomopsis species


The members of the Diaporthe/Phomopsis complex consist of P. longicolla and three varieties of D. phaseolorum, in which D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis cause northern and southern stem canker of soybean, respectively, while D. phaseolorum var. sojae and P. longicolla cause pod and stem blight (Vidi et al., 2011). However, P. longicolla primarily causes agent of soybean seed decay, while other Diaporthe/Phomopsis species may be also associated with this disease. All members of this complex can also colonize other plants, but only on soybean they cause economically important damage. Systematic position. Domain Eukaryota, kingdom Fungi, subkingdom Dikarya, phylum Ascomycota, subphylum Pezizomycotina, class Sordariomycetes, subclass Sordarimycetidae, and order Diaporthales. Teleomorph Diaporthe was placed in the family Diaporthaceae, while its anamorph Phomopsis belongs to the family Valsaceae. Symptoms. D. phaseolorum var. caulivora and D. phaseolorum var. meridionalis cause typical symptoms of stem canker. The initial symptoms are reddish-brown lesions limited by black border that appear on the lower nodes of soybean stem (Fig. 12). These lesions can develop into elongated, sunken, dark brown cankers that spread up and down along the stem. Stem tissue inside the lesions becomes necrotic, the transport of water and nutrients is interrupted, and the infected plants wilt and dry up prematurely. The difference between symptoms of northern and southern stem canker is that within the lesions caused by D. phaseolorum var. meridionalis formed pycnidia are arranged in parallel lines.

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Figure 12. Symptoms of (A) northern and (B) southern stem canker

Symptoms of pod and stem blight of soybean occur on all aboveground parts of plants, but they are considerably less frequent on leaves. First disease symptoms can be seen on the stem or branches, typically occurring around damaged and injured parts of the stem. These areas are covered with numerous black dot-like pycnidia which are arranged in parallel lines (Vidi et al., 2011). During time, the stem can be completely covered by pycnidia (Fig 13). Similarly, the pods are also covered with pycnidia, which are not arranged in lines, but in the form of scattered black dots. Based on the described symptoms it is not possible to distinguish D. phaseolorum var. sojae from P. longicolla (Vidi et al., 1995; 1998). Phomopsis seed decay (PSD) is the major cause of poor seed quality in most soybean-growing countries. The agents of PSD favour long rainy and warm periods during soybean maturation and harvest. Infected seeds are usually small, shrunken, with cracked seedcoats and often appear chalkywhite (Fig. 13). The less infected seeds have a normal appearance, without signs of the disease (latent infection). They have reduced germinability, vitality and quality.

Figure 13. Symptoms of (A) pod and stem blight and (B) Phomopsis seed decay.

Morphology. D. phaseolorum is endophytic homothallic fungus. On PDA, all varieties of D. phaseolorum produce white aerial mycelium that changes colour with age. Within the mycelium, individual small and black stromatic structures are formed. D. phaseolorum var. caulivora rarely forms individual pycnidia on the surface of the colony, while D. phaseolorum var. sojae always forms unilocular and multilocular pycnidial conidiomata within the stroma. Pycnidia of D. phaseolorum var. caulivora are without necks and produce only alpha conidia, while pycnidia of D. phaseolorum var. sojae have short necks or without it, and produce alpha and beta conidia. Within the stroma, both varieties form perithecia with a large number of asci, each containing eight ascospores which initiate primary infections (Fig. 14). 356

P. longicolla differs from other Diaporthe/Phomopsis species in its morphology (Fig. 14). Aerial mycelium is white, but around centre a yellow-green ring forms. The fungus forms very large stromatic structures in which forms multilocular and unilocular pycnidial conidiomate. The conidiomate have very long necks that release the alpha and beta conidia. This fungus does not have a perithecial stage.

Figure 14.  Colony and morphological characteristics of (A) D. phaseolorum var. caulivora, (B) D. phaseolorum var. sojae and (C) P. longicolla.

Physiological specialization. D. phaseolorum species complex consists of several varieties: var. phaseolorum, var. batatatis, var. sojae, var. caulivora, and var. meridionalis (MycoBank, 2012). These varieties have been distinguished basis on the morphological characteristics, pathogenicity, host range, and geographic origin. The var. phaseolorum occurs only on lima beans (Phaseolus lunatus) and causes pod blight, while var. batatatis caused dry rot only on sweet potato (Ipomoea batatas). The other three varieties are described primarily on soybean, but also occur on other legumes and non-leguminous hosts. Studies of the pathogenicity of a large number of D. phaseolorum var. caulivora isolates, observed variability in the parasite and based on this it was assumed that there are more races. Significant differences have been shown in the reaction of some soybean cultivars to isolates originating from the north compared to isolates from the south of USA. Based on the reaction of six soybean cultivars the same number of physiological races of the fungus were differentiated (Keeling, 1984). The stem canker was divided into northern and southern which were named caulivora and meridionalis, respectively; first as formae speciales (Morgan-Jones, 1989) and then as varietas (Fernndez & Hanlin, 1996) of D. phaseolorum.

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