Microbial Plant Pathogens: Detection and Management in Seeds and Propagules

By Perumal Narayanasamy

Healthy seeds and propagules are the basic requirement for producing good grains, fruits and vegetables needed for human survival and perpetuation. Dispersal of microbial plant pathogens via seeds and propagules has assumed more importance than other modes of dispersal, as infected seeds and propagules have the potential to become the primary sources of carrying pathogen inoculum for subsequent crops. Several diseases transmitted through seeds and propagules have been shown to have the potential to damage economies as a result of huge quantitative and qualitative losses in numerous crops. Hence, it is essential to rapidly detect, identify and differentiate the microbial plant pathogens present in seeds and propagules precisely and reliably, using sensitive techniques.

Microbial Plant Pathogens: Detection and Management in Seeds and Propagules provides a comprehensive resource on seed-borne and propagule-borne pathogens. Information on the biology of microbial pathogens, including genetic diversity, infection process and survival mechanisms of pathogens and epidemiology of diseases caused by them, are discussed critically and in detail to highlight weak links in the life cycles of the pathogens.
Development of effective disease management systems, based on the principles of exclusion and eradication of pathogens and immunization of crop plants to enhance the levels of resistance of cultivars to diseases, has been effective to keep the pathogens at bay. The need for production of disease-free seeds/propagules has been emphasized to prevent the carryover of the inoculum to the next crop or introduction of the pathogens to other locations. Effectiveness of adopting simple cultural practices and development of cultivars resistant to diseases through traditional breeding methods or biotechnological approach have resulted in reducing the pathogen inoculum and disease incidence. Although application of different chemicals may reduce the disease incidence effectively, biological management of crop diseases, employing potential biological control agents have to be preferred to preserve the agroecosystems. Greater efforts have to be made to integrate compatible strategies to enhance the effectiveness of diseases management systems.
Protocols appended at the end of relevant chapters form a unique feature of this book to enable the researchers to fine-tune their projects.

This 2 volume set provides comprehensive and updated information about the economically-important groups of microbial plant pathogens carried by seed and propagules. Graduate students, researchers and teachers of plant pathology, plant protection, microbiology, plant breeding and genetics, agriculture and horticulture, as well as certification and quarantine personnel will find the information presented in this book useful.

• Language: English
• Publisher: Wiley
• Release date: Dec 12, 2016
• ISBN: 9781119195795

Book preview

Table of Contents

Cover

Volume 1

Title Page

Preface

Acknowledgement

Volume 1: Pathogen Detection and Identification

1 Introduction

1.1 Concepts and Implications of Pathogen Infection of Seeds and Propagules

1.2 Economic Importance of Seed‐ and Propagule‐Borne Microbial Pathogens

1.3 Nature of Seed‐ and Propagule‐Borne Microbial Pathogens

1.4 Development of Crop Disease Management Systems

References

2 Detection and Identification of Fungal Pathogens

2.1 Detection and Differentiation of Fungal Pathogens in Seeds

2.2 Detection and Differentiation of Fungal Pathogens in Propagules

2.3 Appendix

References

3 Biology of Fungal Pathogens

3.1 Biological Characteristics

3.2 Physiological Characteristics of Fungal Pathogens

3.3 Genotypic Characteristics of Fungal Pathogens

3.4 Influence of Storage Conditions

3.5 Appendix

References

4 Process of Infection by Fungal Pathogens

4.1 Invasion Paths of Seedborne Fungal Pathogens

4.2 Invasion Paths of Propagule‐Borne Fungal Pathogens

References

5 Detection and Identification of Bacterial and Phytoplasmal Pathogens

5.1 Detection and Identification of Bacterial Pathogens

5.2 Detection of Bacterial Pathogens in Propagules

5.3 Detection of Phytoplasmal Pathogens

5.4 Appendix

References

6 Biology and Infection Process of Bacterial and Phytoplasmal Pathogens

6.1 Biology of Bacterial Pathogens

6.2 Disease Cycles of Seedborne Bacterial Pathogens

6.3 Disease Cycles of Propagule‐Borne Bacterial Pathogens

6.4 Biology of Phytoplasmal Pathogens

6.5 Disease Cycles of Phytoplasmal Pathogens

6.6 Appendix

References

7 Detection and Identification of Viruses and Viroids

7.1 Detection of Viruses in Seeds

7.2 Detection of Viruses in Propagules

7.3 Detection of Viroids in Seeds

7.4 Detection of Viroids in Propagules

7.5 Appendix

References

8 Biology and Infection Process of Viruses and Viroids

8.1 Characteristics of Plant Viruses

8.2 Biological Properties of Viruses

8.3 Infection Process of Plant Viruses

8.4 Characteristics of Viroids

8.5 Infection Process of Viroids

References

Index

Volume 2

Title Page

Preface

Acknowledgement

Volume 2: Epidemiology and Management of Crop Diseases

9 Epidemiology of Seed‐ and Propagule‐Borne Diseases

9.1 Epidemiology of Fungal Diseases

9.2 Epidemiology of Bacterial Diseases

9.3 Epidemioloy of Virus Diseases

References

10 Crop Disease Management: Exclusion of Pathogens

10.1 Health Status of Seeds and Propagules

10.2 Plant Quarantines for Preventing Entry of Microbial Pathogens

10.3 Production of Disease‐Free Seeds and Propagules

10.4 Appendix

References

11 Crop Disease Management: Reduction of Pathogen Inoculum

11.1 Reduction of Pathogen Inoculum by Cultural Practices

11.2 Reduction of Pathogen Inoculum by Physical Techniques

11.3 Reduction of Pathogen Inoculum by Chemical Techniques

References

12 Crop Disease Management

12.1 Types of Disease Resistance: Enhancement of Genetic Resistance of Crop Plants

12.2 Identfication of Sources of Resistance to Crop Diseases

12.3 Improvement of Disease Resistance Through Biotechnological Approaches

References

13 Crop Disease Management: Biological Management Strategies

13.1 Evaluation of Biotic Agents for Biological Control Potential

13.2 Evaluation of Abiotic Agents for Biological Control Potential

13.3 Methods of Application of Formulated Products of Biological Control Agents

13.4 Integration of Biological Control with Other Management Practices

References

14 Crop Disease Management: Chemical Application

14.1 Application of Fungicides

14.2 Application of Chemicals Against Bacterial Diseases

14.3 Application of Chemicals Against Virus Diseases

References

15 Crop Disease Management: Integration of Strategies

15.1 Development of Integrated Disease Management Systems

15.2 Management of Fungal Diseases

15.3 Management of Bacterial Diseases

15.4 Management of Virus Diseases

References

Index

End User License Agreement

List of Tables

Chapter 02

Table 2.1 Effect of alkali treatments on the colony diameter and sporulation of seedborne fungi associated with peanut seed lots* (Elwakil et al. 2007).

Table 2.2 Comparative effectiveness of treatments with electrolyzed water (AEW) or sodium hypochloride (NaOCl)a (Bonde et al. 2003).

Table 2.3 DNA contents (pg/µg plant DNA) of Microdochium majus and M. nivale in pooled historical grain samples of wheat and barley during (1957–2000) (Nielsen et al. 2013). From each period, the sample was pooled using one sample per year which was again pooled from four samples in the individual year.

Table 2.4 Quantification of V. dahliae using quantitative polymerase chain reaction (qPCR, mean ± standard deviation) in commercial spinach seed lots (Duressa et al. 2012).

Table 2.5 Variations in in vitro growth, in vitro levels of DON and zearalenone produced, and disease rating of four typed Fusarium isolates compared to the F. graminearum isolates collected in North Carolina (Walker et al. 2001).

Chapter 03

Table 3.1 Assessment of variability in pathogenic potential of fungal pathogens using differential cultivars/genotypes of crop plants.

Table 3.2 Average spread in wheat spikelets and toxin accumulation in inoculated spikelets in cultivar Norm after inoculation with NIV‐ or DON‐type isolates of F. graminearum clade from Lousiana (A) or NIV‐type isolates of F. graminearum and F. asiaticum from Louisiana (B) (Gale et al. 2011).

Chapter 04

Table 4.1 Invasion paths of seedborne fungal pathogens.

Table 4.2 Effects of Fusarium graminearum on disease incidence and mycotoxin accumulation (µg g–1) in grains of seven wheat cultivars (Yoshida and Nakajima 2010): A: Chikugoizumi; B: Norin 61; C: Shiroganekomugi; D: Minaminokaori; E: Saikai 165; and F: Sumai 3.

Chapter 05

Table 5.1 Comparative efficiency of three different laboratory methods in detecting X. campestris pv. undulosa in wheat seeds and influence of seed infestation on seedling infection (Frommel and Pazos 1994).

Table 5.2 Comparative efficacy of MC‐sELISA and ELISA formats in detecting Acidovorax avenae pv. citrulli in seeds of watermelon and cucumber from plants infected by the pathogen (Himananto et al. 2011).

Table 5.3 Colony formation of sorted Cmm cells labeled with Calcein AM, cFDA, PI or combinations of Calcein AM, cFDA with PI, after plating on GNA medium (Chitaara et al. 2006).

Table 5.4 Detection by PCR assays using different target sequences of seedborne bacterial plant pathogens.

Table 5.5 Detection of Xylella fastidiosa (Xf) by PCR and isolation of Xf in culture from sweet orange seedlings obtained from seeds extracted from CVC‐affected fruits (Li et al. 2003).

Table 5.6 Comparative sensitivity of PCR formats determined by using different CFUs of Pseudomonas syringae pv. phaseolicola (Schaad et al. 2007).

Table 5.7 Comparative efficacy of the BX‐S and Aac primer sets in IMS‐real‐time PCR assay for the detection of Acidovorax avenae ssp. citrulli (Aac) in seed lots of watermelon with different levels of infestation (Bahar et al. 2008).

Table 5.8 Comparative efficacy of ELISA, DIG‐labeled PCR, and nested PCR assays for the detection of C. michiganensis ssp. sepedonicus in seed tubers and stem samples of potato (Lee et al. 2001).

Table 5.9 Virulence characteristics of Idaho isolates of Streptomyces on radish in comparison to type strain of S. scabieiT (ATCC 49173) (Wanner 2007).

Table 5.10 Detection of "Ca. Liberibacter asiaticus" in seedlings by qPCR assay at different time intervals between planting seed and DNA extraction and testing (Hartung et al. 2010). Total number of tests (1216) performed from January 2006 to December 2006.

Table 5.11 Influence of temperature range on zebra chip disease development in inoculated potato plants (Munyaneza et al. 2012).

Table 5.12 Comparative efficacy of diagnostic techniques in detecting L. xyli ssp. xyli infection in sugarcane leaf 5 (fourth leaf basal to leaf 1; fully expanded youngest leaf) of sugarcane stalks (7 month old) (Grisham et al. 2007).

Chapter 06

Table 6.1 Quantification of "Ca. Liberibacter asiaticus" genomes in DNA extracts of fruit tissues of huanglongbing (HLB‐) infected citrus plants using qPCR assay (Li et al. 2009a).

Table 6.2 Influence of temperature on development of ZC symptom and pathogen growth (potato plants inoculated with ZC pathogen and maintained in growth chamber at different temperatures) (Munyaneza et al. 2012).

Chapter 07

Table 7.1 Plant viruses belonging to different families, genera, and type species transmitted through seeds/propagules of infected plants (Mayo and Brunt 2007).

Table 7.2 Seed transmission of plant viruses in different host plant species, as revealed by immunoassays.

Table 7.3 Infectivity of RYMV in extracts from seeds of rice and wild host species as determined by ELISA at two stages (Allarangaye et al. 2006).

Table 7.4 Confirmation of ELISA results by vascular puncture inoculation of corn plants (Forster et al. 2001).

Table 7.5 Detection of Plum pox virus‐M in whole seeds and seed parts (Milusheva et al. 2008).

Table 7.6 Viruses detected in propagules of crops using immunoassays.

Table 7.7 Comparative efficiency of the polymerase chain reaction (PCR) and grafting for detection of geminiviruses in sweet potato plants (Li et al. 2004).

Table 7.8 Comparison of levels of sensitivity and specificity of methods for detection of Plum pox virus (Capote et al. 2009).

Table 7.9 Viroids belonging to different families and genera transmitted through seeds/propagules of infected plants (Mayo and Brunt 2007).

Chapter 10

Table 10.1 Effect of heat treatments on survival of strawberry plants infected by angular leaf spot disease under field conditions (Turechek and Peres 2009).

Chapter 14

Table 14.1 Effect of timings of fungicide application at anthesis on infection parameters and mycotoxin accumulation in wheat cultivar Norin 61 in 2006 (Yoshida et al. 2012).

Table 14.2 Effect of treatment with PA in combination with different seed drying temperatures on seed transmission of BFB in watermelon (Hopkins et al. 2003).

List of Illustrations

Chapter 02

Figure 2.1 Sorting of wheat kernels damaged by Fusarium spp., using high‐speed optical sorter (Scam Master II DE). Rapid separation of Fusarium damage kernels (FDK) with different concentrations of deoxynivalenol (DON).

Figure 2.2 Effectiveness of acidic electrolyzed water (AEW) and sodium hypochlorite (NaOCl) in eliminating the wheat kernel bunt pathogen Tilletia indica. Note the inhibition of development of the pathogen in the medium amended with seed washes obtained after treatment with AEW and NaOCl.

Figure 2.3 Detection of Colletotrichum lindemuthianum in infected bean seed powder containing different concentrations of the pathogen, using nested PCR assay. Lanes: M‐100‐bp DNA ladder; 1, 100%; 2, 80%; 3, 60%; 4, 40%; 5, 20%; 6, 10%; 7, 8%; 8, 6%; 9, 4%; 10, 2%; 11, 1%; 12, 0%; 13, anthracnose‐resistant genotype G2333; 14, negative control (water); and 15, positive control (pathogen DNA).

Figure 2.4 Estimation of biomass of Septoria tritici in inoculated wheat leaves, after different periods of incubation at 12°C and 18°C employing PCR/PicoGreen assay.

Figure 2.5 Detection of Verticillium longisporum in spinach seed predominantly infected by V. dahliae. Note the presence of 340 bp amplicon unique to V. longisporum detected by PCR assay. Lane 1: DNA template extracted from a pure culture of V. longisporum strain Bob 70; lane 2: DNA template of V. longisporum strain Bob 70 in seed DNA background; lane 3: DNA templates extracted from V. dahliae strain VdLs 16; lanes 4–18: templates expressed in seed lots; lane 19: negative control (water); lane L: DNA standards.

Figure 2.6 Assessment of DNA contents of Sclerotinia sclerotiorum present along with Botrytis cinerea in mixed populations using quantitative PCR assay. Bars indicate the standard errors of means.

Figure 2.7 Differentiation of isolates of Fusarium graminearum, F. avenaceum and F. culmorum based on random amplified polymorphic DNA (RAPD) profiles formed by Operon primer OPB15. Lane 1: Molecular standards; lane 2: F. avinaceum; lane 3: F. culmorum (R‐6565); Lanes 4 and 5: F. graminearum (R‐6914 and R‐6925) and lanes 6 to 29: isolates 1 to 24 of F. graminearum from North Carolina, USA.

Figure 2.8 Detection of Tilletia indica, T. walkeri and T. horrida by PCR assay using T. indica‐specific primer pairs Ti17M/M2 and Ti57M1/M2. Note products of similar size produced from DNA of T. indica and T. walkeri, but not from T. horrida. Lanes 1 and 14: Molecular ladder; lanes 2, 3, 6, 7, 10 and 11: T. indica; lanes 4, 8 and 12: T. walkeri and lanes 5, 9 and 13: T. horrid.

Figure 2.9 Differentiation of isolates of T. indica, T. walkeri, T. horrida and T. barclayana based on the sequence analysis of ITS region ScaI restriction enzyme digest of ITS, 5.8S and ITS ribosomal DNA PCR products differentiated by agarose gel electrophoresis. Lanes 1 and 20: DNA ladder; lanes 2–7: T. indica; lanes 8–13: T. walkeri; lanes 17–19: T. barclayana.

Chapter 03

Figure 3.1 Determination of DNA contents of Ramularia collo‐cygni in different layers of leaves and ears of barley cultivar Lanark 2009 at different sampling dates, using quantitative assay.

Figure 3.2 Assessment of virulence and toxigenic potential of isolates representing genetic variation in Fusarium graminearum present in Louisiana State, USA. Gray columns: average spread in spikelets in point‐inoculated wheat spikes of susceptible cultivar Norm. Black columns: average trichothecene mycotoxins accumulation in ppm in inoculated spikelets; Fg: Fusarium graminearum; GC: Gulf coast population of F. graminearum; Fa: F. asiaticum; NIV: nivalenol; DON: deoxynivalenol.

Chapter 04

Figure 4.1 Response of chasmogamous (open‐flowering) and cleistogamous (closed‐flowering) barley to inoculation with Fusarium graminearum at different days after anthesis (daa). (a) Spikes at 2 daa; (b) spikes at 8 daa; (c) spikes at 10 daa; (d) view of ventral side of floret at 6 daa; and (e) 9 daa. Until 6 daa, anthers remained within closed florets; most anthers were partially extruded at 8 daa and anthers were entirely extruded at 10 daa. Arrows indicate extruded anthers.

Figure 4.2 Effects of inoculation of two barley cultivars (1) Nishinochikara and (2) Minorimuge on the appearance of mature grains, following inoculation with Fusarium graminearum at different days after anthesis (daa). (a) Unioculated; (b) inoculated at anthesis; (c) inoculated at 10 daa; and (d) inoculated at 20 daa. Mycotoxins contents (DON + NIV) ranged from 0.0 to 9.0 µg g–1 in Nishinochikara samples and from 0 to 10.3 µg g–1 in Minorimuge samples.

Figure 4.3 Visualization by light microscopy of pathogen invasion in floral parts of rice spikelets inoculated with Ustilaginoidea virens. Note infection of three special filaments located between ovary and lodicules near the lemma in semithin sections. (a, b) Floret filled with mass of dense pathogen hyphae; arrows show location of three infected filaments; and (c) shrunken uninfected filaments (F1) surrounded by dense hyphal mass.

Figure 4.4 Transmission electron microscopic visualization of infection process of Ustilaginoidea virens in infected rice lodicules. Note the presence of (a): pathogen hyphae (PH) in firm contact with lodicules; (b) and (d): intracellular colonization in the outer layers of cells of lodicules and (c): light microscopic examination showing hyphae (arrowed) extending into intracellular space, but not reaching the vascular tissues.

Chapter 05

Figure 5.1 Isolation of Clavibacter michiganensis subsp. michiganensis (Cmm) from tomato seed extracts on YPGA medium at different concentrations before and after immunomagnetic separation (IMS). Left: saprophytic bacterial populations at high levels and right: effective reduction of saprophytic bacterial population by IMS, favoring the development of Cmm.

Figure 5.2 Detection of Xanthomonas campestris pv. vesicatoria (Xcv) by immunogold electron microscopy using the monoclonal antibody MAB 4AD2 specific to the pathogen. Note the uniform distribution of immunospecific epitopes on the cell wall of Xcv.

Figure 5.3 (a) Visualization of "Candidatus Liberibacter asiaticus" in the transverse sections of phloem sieve element in seed coat of grapefruit cultivar Conners using transmission electron microscopy (TEM). B: bacterial cells; SP: sieve plate, and M: mitochondria (b) Cross‐section of phloem sieve element showing large number of bacterial cells.

Figure 5.4 Detection of Xanthomonas campestris pv. campestris (Xcc) in Brassica seed using multiplex PCR assay targeting sequences of hrpF gene. (a) Multiplex reactions for amplification of pathogen‐specific 619 bp and 360 bp products from Brassica samples and (b) pathogen‐specific hrpF product of 619 bp amplified from X. campestris.

Figure 5.5 Detection of Xylella fastidiosa (Xf) in citrus fruits by polymerase chain reaction (PCR). Note the presence of pathogen‐specific 472 bp amplicon (arrow) in different fruit tissues. Lane 1: peduncle; lane 2: exocarp; lane 3: mesocarp; lane 4: endocarp; lane 5: septa; lane 6: central axis; lane 7: Xf strain 9a5c; lane 8: central axis of healthy fruit (negative control). DNA ladder (100 bp) is positioned at the extremities.

Figure 5.6 Detection of Xylella fastidiosa (Xf) by PCR assay in different seed tissues and emerging seedlings, as revealed by Xf‐specific product of 472 bp (arrow). DNA ladder (100 bp) is positioned at the extremities. Lane 1 and 11: Xf strain 9a5c DNA (positive controls); lane 2: seed coat; lanes 3 and 4: embryos; lanes 5–7: in vitro isolates from cultivars Valencia, Natal, and Pera; lanes 8–10: seedlings of cultivars Pera, Natal, and Valencia; lane 12: water (negative control).

Figure 5.7 Detection of "Candidatus Liberibacter asiaticus" in greenhouse‐grown sweet orange trees inoculated by bud grafting. Monitoring pathogen distribution by determining populations of pathogen (genomes) in bark tissues of stem and root at different positions by qPCR assays.

Figure 5.8 Determination of detection threshold of Pseudomonas phaseolicola pv. phaseolicola, using standard PCR and nested PCR formats. Nested PCR assay was more sensitive than standard PCR assay. Ethidium bromide‐stained bands (white on black background, upper panel) and bands detected by Southern hybridization (black bands on white background, lower panel) represent pathogen DNA concentrations in a descending order; two left lanes in each group correspond to duplicate PCR reactions with external primers and two right lanes to duplicate reactions with nested primers.

Figure 5.9 Detection of Acidovorax avenae subsp. avenae by nested PCR assay in spiked rice seed samples (a) without enrichment and (b) with enrichment in SP liquid medium. Note the presence of amplified product of 224 bp from pathogen DNA at different concentrations. Lane 1: (1–2) × 10⁴; lane 2: (1–2) × 10³; lane 3: (1–2) × 10²; lane 4: 10–20; lane 5: 1–2 CFU g–1 of seeds; lane 6: uninoculated seed extract.

Figure 5.10 (a) Relationship between populations of pectolytic erwinias and seed germination (negative) and (b) relationship between erwinia populations and disease incidence (positive).

Figure 5.11 Comparative efficacies of nested PCR and DIG‐labeled PCR formats for the detection of Clavibacter michiganensis subsp. sepedonicus in field‐grown potato cultivars. (a) Nested PCR products on agarose gel (1%); Rus: Russet Burbank; Nor: Norchip; T1–T4: tuber samples; S1–S4: stem samples; lane S: DNA standard (1 kb) and C‐w: water control. (b) DIG‐labeled PCR products dot‐blotted on nylon membrane detected using NBT and BCIP. Row a: samples Rus T1–T3, Red T1, Red T2, and Nor T1; row b: samples Nor T2–T4 and Rus S1–S3; row c: samples Rus S4 and C‐w.

Figure 5.12 Assessment of genotypic diversity of eleven strains of Xanthomonas citri subsp. citri using BOX‐, ERIC‐, and rep‐PCR assays. M: molecular size standards. Genomic profiles were separated on agarose gels and stained with ethidium bromide.

Figure 5.13 Detection of Clavibacter xyli subsp. xyli (Cxx) in sugarcane vascular samples by PCR assay and Southern blotting. (a) PCR products were stained with ethidium bromide; lane 1: DNA standards; lane 2: cultured pathogen cells; lane 3: water control; lanes 4–23: sugarcane vascular samples. (b) Autoradiograph of gels in panel (a) probed with ³²P labeled L1/G1, primed PCR product from Cxx genomic DNA.

Figure 5.14 Detection and identification of Idaho Streptomyces isolates with three primer sets for amplification of the same set of DNA templates. (a) Primers ASE3/Scab2m producing 474 bp fragment; (b) primers ASE3/Aci2 producing 472 bp fragment; and (c) primers Aci1/Aci2 producing 1278 bp fragment. Lane M: DNA ladder; lane 1: S. scabies ATCC49173; lane 2: IDOI‐16c (S.europaiscabiei); lane 3: S. acidiscabiesT ATCC49003; lane 4: S. acidscabies ME02‐6987A; lane 5: IDOI‐6.2A; lane 6: IDOI‐12c; lane 7: ID03‐1A; lane 8: ID03‐2A; lane 9: ID03‐3A.

Figure 5.15 Detection of huanglongbing (HLB) infection by biological tests on sour orange seedlings S389, S433, and S391 from left, and healthy sour orange seedlings of the same age on the right.

Figure 5.16 Detection of two strains of Ralstonia solanacearum by PCR using two primer sets AKIF‐AKIR and 2IF‐2IR: (a) MAFF 211490 and (b) MAFF 211471. Lanes 1 and 2: 2.2 × 10⁴ CFU; lanes 3 and 4: 2 × 10³ CFU; lanes 5 and 6: 6.2 × 10² CFU; lanes 9 and 10: 2 CFU; lane 11: control without template; lane m: DNA ladder marker (100 bp).

Figure 5.17 Detection of Clavibacter michiganensis subsp. sepedonicus by PCR assay performed at two melting temperatures of 85.5°C (positives) and 94.5°C (negatives). Left: DNA size standards; lanes 1–5: negative samples; lanes 6–8: positive samples generating a 152 bp amplicon.

Figure 5.18 Optimization of real‐time PCR assay with primer pair VM3/4 to illustrate sensitivity and linearity of the assay using 10‐fold serial dilutions of Xanthomonas citri pv. aurantifolii B DNA. (a) Sensitivity of the real‐time PCR assay; and (b) standard curve showing linear relationship between cycle number and pathogen concentration.

Figure 5.19 Detection of Xanthomonas citri (Xc) 3213 by real‐time PCR assay in herbarium samples collected in 1912. Lanes 1 and 2: A1 extract and DNA; lanes 3 and 4: A2 extract and DNA; lanes 5 and 6: F3 extract and DNA; lanes 7 and 8: F4 extract and DNA; lane 9: water control; lane 10: 10 ng of Xc 3213 DNA; lanes 11–14: Kingsley’s primer pair PCR products; lane 11: A1 DNA; lane 12: A2 DNA; lane 13: F3 DNA; lane 14: F4 DNA; lane 15: water control; lane 16: 10 ng of X. citri pv. citri A DNA; lane M: DNA marker standards.

Figure 5.20 Detection of Xanthomonas axonopodis pv. citri (Xac) by isolation, standard PCR, and real‐time PCR assays in citrus fruit lesions. Bars represent percentages of positive detections by different detection methods.

Figure 5.21 Patterns of hybridization of DIG‐labeled PCR amplicons from Erwinia carotovora subsp. atroseptica (Eca), E. chrysanthemi (Ec), and Clavibacter michiganensis subsp. sepedonicus (Cms) on the oligonucleotides array. a: Eca strain 31; b: Ec strain 340; and c: Cms strain R3. Positive hybridization signals are seen as dark spots within template circles.

Figure 5.22 Patterns of hybridization of DIG‐labeled PCR amplicons from a mixture of E. carotovora subsp. atroseptica (Eca) and E. chrysanthemi (Ec) on the oligonucleotides array. (a) Mixture of Eca strain 31 and Ec strain 340; (b) DNA from Eca‐inoculated potato tuber tissues; and (c) DNA from potato tissue culture. Positive hybridization signals are observed as dark spots within template circles.

Figure 5.23 Amplification curves of fluorescent signals from different isolates of "Candidatus Phytoplasma mali (AT1), Ca. P. prunorum (ESFY1and ESFY2), and Ca. P. pyri" (PD1) following real‐time PCR. Top: qAP‐16S‐F/R primers; bottom: AP‐MGB probes at 64°C as hybridization temperature.

Figure 5.24 Differentiation of strains of Flavescence dorée (FD) phytoplasma in Serbia using restriction fragment length polymorphism analysis. (a) FD0f3/r2 amplicons; (b) rp(V)F2/rpR1 amplicons of FD‐infected Serbian sample (A10). Fragment sizes from top to bottom: 310, 281, 271, 234, 194, 118, and 72.

Figure 5.25 Detection of potato purple top phytoplasma by real‐time PCR assay. Eight potato plants showing positive reaction in nested PCR assay were analyzed along with a sample from healthy potato plants cultivar Shepody.

Figure 5.26 Patterns of RFLP obtained following digestion of amplicons from "Ca. Phytoplasma mali" isolates with (a) FauI and (b) HpaII restriction enzymes. (a) Isolates 246, 147, 230, T‐4, T‐11, 221, 239, 241, AP (AT‐1 subtype), T‐9, T‐16 (AP‐15 subtype), and T‐10 (AT‐2 subtype). (b) Pattern 1 isolates: 243, 244, 246, 45, 147, 230, T‐4, T‐11, 221, 241, and AP (AT‐1 subtype); pattern 2 isolates: T‐9, T‐16 (AP‐15 subtype), T‐10 (AT‐2 subtype), and AP (AT‐1 subtype). M: DNA size markers.

Figure 5.27 Comparative sensitivities of PCR assays using different primers amplifying different amplicons from the DNA of Spiroplasma citri. (a) Primer P89f/r producing 707 bp product; (b) primer P58‐6f/4r producing a 450 bp product; and (c) Sprialin‐f/r (Spln) producing 675 bp product. Lanes 1 and 9: DNA size standards; lanes 3–7: serial dilutions of S. citri DNA (10–1, 10–2, 10–3, 10–4, and 10–5); lane 8: water control.

Chapter 06

Figure 6.1 Detection of Xanthomonas campestris pv. vitians (Xcv) by isolation from stab‐inoculated lettuce and visualization of phloem and xylem from which the pathogen could be isolated. Isolation of Xcv from stem sections taken at different locations: (a) 2–4 cm at 4 h postinoculation (hpi); (b) 0–2 cm from point of inoculation at 12 hpi; (c) 4–6 cm from point of inoculation at 16 hpi; (d) 0–2 cm from uninoculated plant (control); (e) cross‐section of lettuce stem showing phloem and xylem under light microscope.

Figure 6.2 Relative populations of Pseudomonas fuscovaginae isolated from rice seeds with or without treatment with sodium hypochlorite (NaOCl). Populations of P. fuscovaginae expressed as CFU/100 seeds with different levels of seed discoloration in cultivar Amaroo.

Figure 6.3 Correlation between seed contamination levels and severity ratings of tomato bacterial canker disease. Contamination levels of tomato seeds by Clavibacter michiganensis subsp. michiganensis measured as CFU g–1 of seed and number of diseased seedlings infected per 200 seeds show high positive relationship (r² = 0.9448).

Figure 6.4 Transmission of latent Xanthomonas campestris pv. musacearum (Xcm) in bunch tissues to subsequent generations up to F3 in banana cultivars Pisang Awak and Mbwazirume.

Figure 6.5 Detection of "Candidatus Liberibacter solanacearum" in potato plants exposed to liberibacter‐infective psyllids at 24–28°C by PCR assay. Lane 1: DNA standards; lanes 2–8: liberibacter‐inoculated plants maintained at 12–17°C; lanes 9–15: liberibacter‐inoculated plants maintained at 0–25°C; lanes 16–21: liberibacter‐inoculated plants maintained at 27–32°C; lanes 22–29: liberibacter‐inoculated plants maintained at 32–35°C; lanes 31–37: liberibacter‐inoculated plants maintained at 35–40°C; lane 38: negative control; and lanes 30 and 39: pathogen DNA (positive controls).

Figure 6.6 Symptoms of common scab disease caused by Streptomyces scabiei on potato tubers of cultivar Desiree at harvest, following inoculation at 13 and 18 days after tuber initiation.

Chapter 07

Figure 7.1 Detection of Soybean mosaic virus (SMV) by dot‐immunobinding assay (DIBA) and tissue‐print immunoassay (TPIA) using NBT/BCIP as substrate. (a) DIBA: F3: negative control; F5: positive control. (b) TPIA: A1 and B1: positive controls; H4: negative control. Development of color indicates the presence of SMV in sample extracts.

Figure 7.2 Simultaneous detection of six viruses by tissue‐printing hybridization technique using nonisotopic polyprobe‐6. (a) Positive reactions are indicated by development of color in samples infected by Cucumber mosaic virus, Pepino mosaic virus, Parietaria mottle virus, Potato virus Y, Tomato mosaic virus, and Tomato spotted wilt virus; lack of reaction in healthy samples N‐INF1, N‐INF2, and N‐INF3. (b) Positive reactions are seen in field‐grown plant samples when infected by any one of the viruses with which polyprobe‐6 can hybridize.

Figure 7.3 Estimation of genomic RNA concentration of two strains of Pea enation mosaic virus EMV1 and PEMV2, using multiplex quantitative real‐time RT‐PCR assay in stipule, whole pea, seed coat, and embryo tissues. Genomic RNA concentrations are expressed as mean normalized accumulation (MNA).

Figure 7.4 Detection of grapevine leafroll viruses by immunosorbent electron microscopy (ISEM) using the bivalent reagent CL‐LR3. The bivalent antibody could trap (a) GLRaV‐1 or decorate (b) GLRaV‐7 and (c) GLRaV‐3; bar = 100 nm.

Figure 7.5 Detection of Grapevine virus A (GVA) in tissue prints and extracts of petiole tissues in Vitis spp. and hybrids. Tissue imprints: Columns A and B; Dot blots: Column C. Samples: 1: 110 Richter; 3: Kober 5BB; 5: Vitis rupestris; 7: V. riparia 8‐ LN 33 (interspecific hybrids of different Vitis spp.); 28: Interspecific hybrid Prim (Palatina); 30: V. vinifera culitvar Guzal Kara; TA: positive grapevine cultivar Traminer, lower leaves; TB: positive grapevine cultivar Traminer, upper leaves; F10: plasmid.

Figure 7.6 Detection of sweet potato viruses by PCR assays in sweet potato using three different primer pairs: (a) PW285‐1/PW285‐24; (b) SPG1/SPG2; or (c) SPG3/SPG4. Lane 3: an uncharacterized Jamaican isolate infecting sweet potato; lane 4: an uncharacterized Puerto Rican isolate infecting sweet potato; lane 5: Ipomoea leafcurl virus; lane 6: Tomato yellow leafcurl virus; lane 7: Tomato mottle virus; lane 8: Bean golden mosaic virus; lane 9: Cabbage leafcurl virus; lane 10: Squash leafcurl virus; lane 11: Cotton leaf crumple virus; lane 12: Beet curly top virus; lane 13: a potyvirus infecting sweet potato (negative control); lane 14: water control.

Figure 7.7 Detection of purified Potato virus Y added directly at different concentrations to RT reaction mixture by RT‐DIAPOPS procedure. Four columns represent each dilution of PVY in four independent experiments.

Figure 7.8 Correlation between tuber infection by Potato mop‐top virus (PMTV) and incidence of spraing in Scottish seed potato tubers of cultivar Cara.

Figure 7.9 Detection of Sweet potato leaf curl virus (SPLCV) in in vitro plantlets of sweet potato accessions by real‐time PCR assay. Amplification plot from SPLCV: samples spiked with positive control crossed the cycle threshold; the DNA extract from PI 585052 also crossed the cycle threshold.

Figure 7.10 Detection of Plum bark necrosis stem pitting‐associated virus (PBNSPaV) by nested RT‐PCR assay using ASPn1/ASPn2 primer pair. M: molecular size standards; P: positive virus control; H: healthy plant control. (a) Lanes 1–16; (b) lanes 3–18, 11–14; 16: virus positives from symptomatic and asymptomatic plant samples.

Figure 7.11 Detection of Potato yellow vein virus (PYVV), Tomato infectious chlorosis virus (TICV), and Tomato rattle virus (TRV) by the multiplex PCR assay using serially diluted individual and mixed DNA templates. Lanes 1–7, 8–14, and 15–21: PYVV at original 1:10, 1:100, 1:500, 1:1000, 1:2000, and 1:4000. TICV: original, 1:10, 1:100, 1:200, 1:400, 1:800, and 1:1600. Lanes 22–29: serially diluted samples of three mixed cDNA species: original, 1:10, 1:100, 1:500, 1:1000, 1:2000, 1:4000, and 1:8000. M: molecular size standards (100 bp).

Figure 7.12 Comparative levels of sensitivity of DAS‐ELISA, IC‐PCR and IC‐PCR‐ELISA techniques for the detection of Potato virus Y. IC‐PCR‐ELISA was more sensitive than other two techniques tested.

Figure 7.13 Detection of Plum pox virus (PPV) by real‐time RT‐PCR with (a) SYBR Green and (b) TaqMan chemistries, employing four direct sample preparation methods–dilution, spot, tissue‐print, and squash methods–along with healthy GF305 peach seedlings as negative control. Dilution and spot real‐time RT‐PCR methods were slightly more sensitive than tissue‐print and squash methods.

Figure 7.14 Detection of (a) CEVd, (b) CBLVd, (c) HSVd, (d) CDVd, and (e) CBCVd in mixed infections in citrus plants by Northern hybridization with viroid‐specific probes. Lane 2: Fino lemon; lane 4: Common mandarin; lane 6: Tahiti lime; lane 8: Foster grapefruit; lane 10: sour orange; lanes 1, 3, 5, 7, and 9: corresponding healthy control.

Figure 7.15 Detection of six viroids in mixed infections by dot‐blot hybridization assay using DIG‐labeled probes in extracts of pome and stone fruit trees. A1–11: extracts from plants infected by Apple scar skin viroid (ASSVd); B1–11: extracts from plants infected by Apple dimple fruit viroid (ADFVd); C1–11: extracts from plants infected by Pear blister canker viroid (PBCVd); D1–11: extracts from plants infected by Apple fruit crinkle viroid (AFCVd); E1–11: extracts from plants infected by Hop stunt viroid (HSVd); F1–11: extracts from plants infected by Peach latent mosaic viroid (PLMVd); G1–11: extracts from healthy plants. Single probes tested: row 1 (ASSVd); row 2 (ADFVd); row 3 (PBCVd); row 4 (AFCVd); row 5 (HSVd); and row 6 (PLMVd). Polyprobes tested: poly2‐A (ASSVd and ADFVd, row 7); poly2‐B (PBCVd and AFCVd, row 8); poly2‐C (HSVd and PLMVd, row 9); poly4‐AB (ASSVd, ADFVd, PBCVd, and AFCVd, row 10) and poly6 (row 11).

Figure 7.16 Detection of (a) Chrysanthemum stunt viroid (CSVd) and (b) Potato spindle tuber viroid (PSTVd) by real‐time TaqMan assay using viroid‐specific probes. TaqMan amplification plots for specific detection of CSVd and PSTVd.

Figure 7.17 Comparative levels of sensitivities of electrophoresis, Southern blot hybridization, and RT‐PCR dot‐blot hybridization (DBH) for the detection of Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) in citrus. (a, b) electrophoresis; (b, e) Southern blot hybridization; (c, f) RT‐PCR‐DBH. Lanes: M: molecular standards (100 bp); 1–3: serially diluted positives (10–10⁴); 4: negative control without template; 5: RT product; 6: extracted RNA.

Figure 7.18 Detection of Potato spindle tuber viroid (PSTVd) in potato, tomato, and petunia by RT‐PCR assay. M: DNA size standards; lanes 1, 2, and 7: healthy tomato, petunia, and potato; lane 3: dry tomato infected with Cornell severe PSTVd strain; lane 4: freshly infected petunia; lanes 5 and 6: dry tomato infected with PSTVd‐S; lane 8: water control.

Figure 7.19 Comparative sensitivity of RT‐LAMP and Tsutsumi RT‐LAMP assays for detection of Potato spindle tuber viroid (PSTVd) using different dilutions of total potato plant RNA. Dilutions of total RNA: 1 × 10–2; 1 × 10–3; 1 × 10–4; control: water (without template).

Chapter 08

Figure 8.1 Influence of Bean pod mottle virus (BPMV), Soybean mosaic virus (SMV), and mixed infections on the percentage of seeds of soybean lines with mottling symptoms under field conditions in 2001. Mean percentages of mottled seeds (columns) followed by the same letter are not significantly different as per the Student’s test (P = 0.05).

Figure 8.2 Differentiation of Citrus tristeza virus (CTV) isolates in individual and mixed infections of sweet orange plants based on SSCP profiles characteristic of p18 gene. SSCP profiles of mild isolate T425, severe isolates T388 or T305, or a mixture of T425 and T388 or T425 and T305 in the extracts of inoculated plants determined at 3 years after challenge inoculation with T388 or T305 isolates.

Chapter 09

Figure 9.1 Influence of irrigation on development of Phomopsis longicolla (a) leaf, (b) stem, and (c) pod, represented by area under disease progress curve under nonirrigated (NI), post‐flower irrigation (PF), and pre‐ plus post‐flower irrigation (PPF) environments during 2003–2004. Means followed by the same letter within each irrigation environment are not significantly different (P ≰ 0.05).

Figure 9.2 Effects of irrigation on percentage of recovery of Phomopsis longicolla from (a) infected seed, (b) seed germination percentage, and (c) seed hardiness in soybean crop grown in nonirrigated (NI), post‐flower irrigation (PF), and pre‐ plus post‐flower (PPF) irrigation environments. Means followed by the same letter within each irrigation environment are not significantly different (P ≰ 0.05).

Figure 9.3 Differential disease progress curves of late blight disease in pure stands of potato (a) cultivar Sante and (b) cultivar Cara and mixtures with Sante or Cara at three planting densities.

Chapter 10

Figure 10.1 Effectiveness of in vitro thermotherapy coupled with shoot‐tip grafting for elimination of Indian citrus ringspot virus (ICRSV) from citrus plantlets. Note the absence of ICRSV‐specific amplicons in treated plantlets. Lane M: DNA size standards; lanes 1 and 2: positive and negative controls, respectively; lanes 3–6: samples from treated citrus plantlets.

Figure 10.2 Efficiency of hot water treatment (HT) in eliminating Xanthomonas fragariae from strawberry plants. (a) Survival of plants (percentage) after different periods of heat treatment (min) at 44°C and 48°C; (b) average number of runners in survivors after HT; and (c) average number of flower trusses formed in plants exposed to HT.

Figure 10.3 Detection of sweet potato little leaf phytoplasma in in vitro shoot cultures by PCR assay after cryotherapy. (a, b) Lane M: DNA ladder; lane P: positive control; N: negative control; lanes 1–4: presence of c. 1.8 kb product in (a) and absence of this product from (b) indicating the effective elimination of the pathogen in cryotherapy‐treated shoot cultures.

Figure 10.4 (a) Visualization of sweet potato little leaf phytoplasma in TEM cross‐sections of shoot tips taken at different positions of leaf primordial of plantlets after cryotherapy. (b, e) Cross‐sections of leaf primordial closer to the apical dome (AD) show the elimination of the phytoplasma; (c, d, f) cross‐sections of leaf primordial away from AD show partial or lack of effectiveness of cryotherapy; and (c, d, f, g) phytoplasma cells are indicated by arrows.

Chapter 11

Figure 11.1 Development of potato black dot disease on stems, stolons, and roots of three potato cultivars with different levels of resistance in (a) England and (b) Scotland. Disease severity ratings measured on potato cultivar Maris Piper (■), Sante (Δ), and Saxo (X).

Figure 11.2 Effects of different rotation crops during 2000–2006 on incidence of Rhizoctonia canker, black scurf, and common scab diseases in potato. (a) Rhizoctonia canker; (b) black scurf; and (c) common scab. Two‐year rotational crops include barley (BA), canola (CN), millet/rapeseed (RP), green bean (GB), sweet corn (SC), soybean (SY), and potato (PP, control without rotation). Bars with the same letter are not significantly different as per Fischer’s protected least significant difference test (P < 0.05).

Figure 11.3 Effectiveness of mulching with bicolor aluminized polyethylene before planting and application of chemicals provide additive effects by reducing disease severity. Unfilled bars: with chemical (Kocide 2000 + Neemguard) application and filled bars: without chemical application.

Chapter 12

Figure 12.1 Effects of low‐ and high‐titer of Fusarium graminearum conidia in combination with (a) point‐ and (b) spray‐inoculation with the pathogen on (c–d) means of spikes and ears infected by DON‐ and NIV‐producing isolates in spring wheat cultivar Mercia. (e–f) High‐titer inoculum produced greater disease severity; DON‐producing isolates spread rapidly, while NIV‐producing isolate was restricted to inoculated spikelets within the spike.

Figure 12.2 Evaluation of corn hybrids under inoculated conditions for resistance to Fusarium ear kernel rot disease. Assessment of disease severity by visual rating based on percentage (0–100%) of ears with symptoms in different corn hybrids.

Figure 12.3 Disease severity induced by Streptomyces scabiei in resistant clone 65A and susceptible parent Iwa.

Figure 12.4 Reactions of transgenic carrot plants expressing chitinase CHIT36 gene to the fungal pathogens Alternaria dauci, A. radicina, and Botrytis cinerea following inoculation of detached leaflets and petioles. Values represent the percentage of nontransgenic Koral control for transgenic line 96‐183. N176 and N184: the nontransgenic clones not expressing CHIT36; D.c.c: Dacus carota ssp. Commutatus; YEL: yellow leaf mutant; bar: standard error; star: clones with significantly lower values than the control based on Dunnett’s test at P = 0.05 for A. dauci and P = 0.01 for A. radicina and B. cinerea.

Figure 12.5 Northern blot analysis revealing transcription of xa21 gene of rice in Citrus sinensis cultivars (a) Hamlin; (b) Pera; (c) Natal; and (d) Valencia leaves. Lanes 1–8: transgenic lines expressing xa21 mRNA; lane C: nontransgenic plant (control).

Chapter 13

Figure 13.1 Induction of resistance to tomato late blight by application of Pseudomonas fluorescens SS101 or massetolide A to leaves of tomato cultivar Moneymaker and its transgenic derivative nahG. Effect on (a) lesion area (mm²) and (b) disease severity (%) determined at 7 days postinoculation with Phytophthora infestans; asterisk (*) indicates significant reduction in disease severity relative to control (P < 0.05); filled columns: control; blank columns: SS101 strain; lined columns: massetolide A.

Figure 13.2 Effect of BABA application on the development of defense responses in potato cultivars challenged with Phytophthora infestans. Note hypersensitive response (HR‐) like lesions formation in leaves treated with BABA, irrespective of infection at all intervals after inoculation; i: idioblast; m: mesophyll cells; HR: HR‐like lesions in BABA‐treated potato cultivar Ovatio at 48 h postinoculation.

Chapter 14

Figure 14.1 Positive correlation between the percentages of necrotic leaf surface on wheat cultivar Maxyl and the copy numbers of tubulin gene (DNA contents) determined by qPCR assay (F = 0.95). Note the disease progress is proportional to the pathogen development.

Figure 14.2 Comparative abilities of Boscalid‐resistant (BR) mutants and their parental isolates in producing sclerotia on PDA medium at 40 days after inoculation. (a–c) Parental isolates; (d–h) BR mutants.

Figure 14.3 Baseline sensitivity frequency distribution of strains of Xanthomonas oryzae pv. oryzae to zinc thiazole.

Figure 14.4 Effect of application of penicillin G (P) or streptomycin (S) individually or in combination (PS) on populations (cells g–1 of plant tissue) in huanglongbing (HB‐) affected citrus seedlings under greenhouse conditions. Penicillin applied at 1 g L–1 and streptomycin at 100 mg L–1; CK: control (water).

Figure 14.5 Effect of application as trunk injection of combination of penicillin and streptomycin (PS) at different ratios on bacterial pathogen titers (cells g–1 of plant tissues) in huanglongbing (HB‐) affected citrus plants under field conditions. PS‐5: penicillin 5 g + streptomycin 0.5 g/100 mL; PS‐10: penicillin 10 g + streptomycin g/100 mL; PS‐0: water control.

Volume 1

Microbial Plant Pathogens

Detection and Management in Seeds and Propagules

Volume 1

P. Narayanasamy

Former Professor and Head

Department of Plant Pathology

Tamil Nadu Agricultural University

Coimbatore, India

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Names: Narayanasamy, P., 1937– author.

Title: Microbial plant pathogens : detection and management in seeds and propagules / P. Narayanasamy.

Description: Hoboken : John Wiley & Sons, 2017. | Includes bibliographical references and index.

Identifiers: LCCN 2016036177 (print) | LCCN 2016037345 (ebook) | ISBN 9781119195771 (cloth) | ISBN 9781119195788 (pdf) | ISBN 9781119195795 (epub)

Subjects: LCSH: Seed‐borne phytopathogens. | Seed‐borne plant diseases.

Classification: LCC SB732.8 .N27 2017 (print) | LCC SB732.8 (ebook) | DDC 632/.3–dc23

LC record available at https://lccn.loc.gov/2016036177

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Dedicated to the memory of my parents for their love and affection

Preface

Agricultural and horticultural crops are raised by using seeds or propagules whose health status has to be ensured for healthy and high‐quality produce to satisfy the needs of human and animal populations. From time immemorial, seeds and propagules were obtained from selected plants growing in the wild environment, saved to grow plants of subsequent generations. In doing so, the improvement of quality parameters such as appearance, color, aroma, and taste was achieved by selecting plants with desired characteristics. However, due importance was not allocated to select plants with resistance to diseases caused by microbial plant pathogens, resulting in progressive increase in the susceptibility of plants to diseases and phenomenal crop losses which were considered responsible for dreadful famines and untold human suffering. Several diseases transmitted through seeds/propagules have been found to be highly destructive, with the potential to ruin the economy of certain countries. Such critical conditions were primarily ascribed to the failure to select disease‐free seeds and propagules for future generations of crops.

Early detection and precise identification of the pathogen(s) present in seeds/propagules which are involved in a disease(s) occurring at a geographical location constitute the basic strategy for development of effective disease management systems suitable for various agroecosystems. Studies of pathogen biology, the infection process, and epidemiology of crop diseases have highlighted the weak links in the life cycles of microbial pathogens in order to disrupt pathogen development and the progress of disease under field conditions. The principles of crop disease management are essentially based on exclusion, eradication, and immunization and various disease management strategies emerge from these principles. The need to produce disease‐free seeds and propagules to restrict the introduction of pathogens into fields/new locations and subsequent disease spread has been clearly indicated by different investigations. The role of quarantines and certification programs in excluding the introduction of new diseases into a country where the pathogen may be absent or less important has been well realized. The effectiveness of adoption of simple cultural practices in restricting disease incidence and further spread has been indicated in some pathosystems. The development of crop cultivars with built‐in resistance to diseases, the most economical disease management strategy, has been achieved through traditional breeding methods or biotechnological approach and has been shown to be instrumental in keeping many diseases under check. Employing biological control agents is advantageous, since this strategy has been demonstrated to be effective not only in restricting the disease incidence and spread but also in preserving the ecological environments. The application of chemicals, although more effective, must be restricted because of the accumulation of chemical residues in grains and other food materials as well as significant environmental pollution. The integration of compatible disease management strategies has been shown to provide additive effects, resulting in enhancement of effectiveness of the disease management systems; greater efforts have therefore been made to explore the possibility of developing integrated disease management systems for several economically important diseases that need to be tackled more effectively.

This book includes information distilled from over 2500 citations following an extensive literature search, with the aim of providing a comprehensive knowledge of various aspects of microbial plant pathogens transmitted via seeds and propagules, particularly pathogen detection and management of diseases caused by them. Graduate students, researchers, and teachers of Plant Pathology, Plant Protection, Microbiology, Plant Breeding and Genetics, Agriculture and Horticulture, and especially certification and quarantine personnel, will find the information presented in this book useful. Protocols appended at the end of relevant chapters form a unique feature of this book and may assist researchers in fine‐tuning their projects.

P. Narayanasamy

Coimbatore, August 2016

Acknowledgement

The author gratefully expresses his sincere appreciation to his alma mater for being an unlimited source of inspiration in reaching his present position of being able to share his experience and thoughts with those who have contributed to the advancement of science in general and plant pathology in particular. He heartily thanks the staff and students of the Department of Plant Pathology, Tamil Nadu Agricultural University for their assistance in one way or another in improving the usefulness of this book to the audience. The efforts of the author to provide useful information on the selected subject matter in this book have become fruitful thanks to the enormous loving support of his wife Mrs N. Rajakumari whom he thanks profusely. With immense pleasure, the author conveys his appreciation to his family members Mr N. Kumar Perumal, Mrs Nirmala Suresh, Mr T. R. Suresh and Mr S.Varun Karthik for encouraging and uplifting his spirits with their affectionate, amiable attitude and deeds.

Various copyright holders/publishers and authors of research papers have kindly granted permission to use the figures published in different journals, and have been credited at the appropriate pages in this book.

Volume 1

Pathogen Detection and Identification

1

Introduction

Plant diseases are caused by biotic and abiotic stresses. The microbial plant pathogens – oomycetes (fungus‐like), fungi, bacteria, phytoplasmas, viruses, and viroids – form a major group of biotic stresses causing a variety of diseases in various crops grown in different agroecosystems, whereas abiotic stresses are due to adverse environmental conditions which may have an influence both on the plants and the pathogens. The environmental conditions have an important role in the buildup of inoculum and in predisposing the plants to infection by the pathogens to a different extent, depending on the geographical locations where the infected seeds/propagules are to be planted.

1.1 Concepts and Implications of Pathogen Infection of Seeds and Propagules

Use of clean seed and propagules (vegetatively propagated planting materials) is emphasized as the basic step for the commencement of implementation of strategies of crop disease management systems. Seedborne pathogens are not confined to the spermosphere alone and they may also be present in or on the surface of other organs or storage tissues of the plant. Seed pathology is considered a branch of plant pathology, devoted to the detection of microorganisms associated with seeds. That seeds are carriers of microbial plant pathogens, resulting in the infection of developing seedlings, has been known for a long time. The Danish Government Institute of Seed Pathology (DGISP) for developing countries was established in 1967 at Copenhagen by the concerted efforts of Dr Paul Neergaard, who is considered as the Father of Seed Pathology. The term seed pathology was introduced by him during investigations of seedborne pathogens and the diseases caused by them. As the Chairman of the Plant Disease Committee (PDC) of the International Seed Testing Association (ISTA), Dr Neergaard has contributed immensely to the standardization of methods for detection of seedborne fungi. He authored the book Seed Pathology, which contains voluminous information on the various aspects of seed pathology and is the main reference book for all plant pathologists. Seed pathology has developed into an important discipline within plant pathology. The propagules are the seed for the asexually propagated crops, and the importance of propagules carrying pathogens in the incidence and spread of crop diseases has been emphasized by several investigations.

The role of seed‐ and propagule‐borne inoculum in crop disease epidemiology and the indispensability of using disease‐free seeds and propagules for profitable crop cultivation are well realized. Several seed‐ and propagule‐borne pathogens have been shown to be disseminated and perpetuated through various other means. Comprehensive studies are therefore essential to elucidate the host–pathogen interactions, leading to the incidence and spread of diseases caused by seed‐ and propagule‐borne pathogens and consequent substantial losses in quantity and quality of the produce induced by them. Some seedborne pathogens may become soilborne when the infected seeds are planted, and the pathogen present in the soil can infect the plants systemically and infect different tissues en route to the seeds as in the case of Rhizoctonia solani causing rice sheath blight (Narayanasamy 2002). Fusarium graminearum, incitant of the Fusarium head blight (FHB) disease of wheat, develops on the infected plant debris left on the soil after harvest. The inoculum from the infested tissues reaches spikes at the time of anthesis through wind currents (Inch and Gilbert 2003). Verticillium dahliae is seedborne in spinach. The possibility of spinach seeds becoming soilborne and infecting lettuce plants was explored under microplot conditions. Verticillium wilt developed on lettuce following two or three plantings of Verticillium‐infested spinach seeds/plant tissues. Polymerase chain reaction (PCR) assay was employed to detect and confirm the identity of V. dahliae in infected lettuce plants obtained from the microplots. In addition, transmission of a green fluorescent protein (GFP) ‐tagged mutant strain of V. dahliae from spinach to lettuce was demonstrated in greenhouse experiments. The results provided clear evidence that the transmission of V. dahliae introduced through infected spinach seed could be the source of inoculum for Verticillium wilt disease in lettuce (Short et al. 2015).

Seeds and propagules infected by microbial pathogens form the primary sources of inoculum from which they are disseminated by other modes of transmission such as soil, water, or wind. As the environmental conditions have significant effects on the pathogen and the crops, incorporation of epidemiological concepts and the effects of various disease management strategies have to be considered as components of seed pathology. As the propagules are the seed for the vegetatively propagated crops, the propagule‐borne pathogens have to be managed by applying different strategies as in the case of seedborne pathogens. It is logical to widen the scope of research and applications pertaining to the microbial pathogens associated with the seeds and propagules, and to develop management systems for restricting the incidence and spread of the diseases caused by them.

1.2 Economic Importance of Seed‐ and Propagule‐Borne Microbial Pathogens

Microbial pathogens have been demonstrated to be the causes of various crop diseases of great historical and economic importance. A major catastrophe due to potato late blight disease caused by Phytophthora infestans occurred during the 1840s, resulting in the infamous Irish potato famine. The acute food shortage was primarily responsible for the migration of about 1.5 million people from Ireland to other countries (Large 1940). A similar magnitude of human suffering due to another fungal pathogen, Helminthosporium oryzae, ruined the rice crops extensively in India. It has been estimated that about 2 million persons perished due to starvation, because of the exorbitant cost of rice which was beyond the reach of the majority of Indians during that decade (Padmanabhan 1973). These two pathogens, transmitted through infected potato tubers and rice grains respectively, revealed the economic importance and social relevance of the pathogens which are either seed‐ or propagule‐borne. Fusarium head blight (FHB) disease infecting wheat and barley is caused by different Fusarium spp. and still remains an enigma for all involved in cereal production. FHB disease is considered as a re‐emerging disease of devastating impact on cereal grain production. A series of severe FHB epidemics that occurred in the USA and Canada during 1991–1996 led to unparalleled economic and sociological impact in the Northern Great Plains region. The combined direct and secondary economic losses between 1993 and 2001 were estimated to cost US $7.7 billion in nine states of USA (Nganje et al. 2004; McMullen et al. 2012). In addition, the fungal pathogens involved in FHB disease produce different kinds of mycotoxins capable of causing ailments in human beings and animals if contaminated grains and feed are consumed. The quantitative and qualitative losses are very difficult to estimate precisely.

Rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae is widespread in all SE Asian countries. In Japan, rice crops in 300,000–400,000 ha were infected by BLB disease with yield losses ranging over 20–30% (Ou 1972). The estimated yield losses due to BLB disease in tropical Asia may vary over 2–74% depending on the location, season, weather, cultivar, and stage of crop growth at the time of infection. Yield loss induced at the kresek phase of the disease was found to be greater in India (Srivastava and Kapoor 1982; Reddy 1989). The bacterial pathogen Xanthomonas axonopodis pv. citri causing citrus canker disease was considered to have been introduced into the USA from Asian countries through infected budwood materials. Quarantine laws were imposed as soon as the first incidence of citrus canker was observed, and the massive eradication of 20 million citrus trees at a cost of US $20 million effectively checked the spread of the disease (Schumann 1991). Tomato bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis is considered to be one of the most destructive bacterial diseases in tomatoes with the potential to cause heavy yield losses. In Ontario, Canada yield losses of up to 84% in commercial fields were recorded (Poysha 1993). In Michigan State, USA tomato bacterial canker disease accounted for as much as US $300,000 in a single year for individual processing tomato growers (Hausbeck et al. 2000). Because of the potentially destructive nature of the pathogen, the Good Seed and Plant Practices Organization functioning in the Netherlands and France directed disease management strategies for preventing infection of tomato seeds and avoiding the use of infected seedlings (de León et al. 2011; Sen et al. 2015). Huanglongbing (HLB), also referred to as greening of citrus caused by Candidatus Liberibacter asiaticus, has been known to affect citrus production in East Asia for more than a century (Bové 2006); the disease is now found in threatening proportions in the USA and other countries. Tree decline, premature fruit drop and formation of small misshapened fruits lead to drastic production losses, incurring an estimated loss of US $3.63 billion in revenue in Florida alone (Hodges and Spreen 2013; Wang and Trivedi 2013). The HLB disease can be spread through infected nursery stock and the Asian citrus psyllid Diaphorina citri, indicating the need for directing management strategies toward production of certified pathogen‐free nursery stock, eradication of infected trees, and reduction of vector populations through chemical application (Gergerich et al. 2015).

The socioeconomic consequences of seedborne viruses such as Bean common mosaic virus (BCMV) and Lettuce mosaic virus (LMV) have been revealed by several investigations (Stewart and Reddick 1917; Zink et al. 1956; Grogan 1983). The number of seedborne viruses reported increased from 47 in 1969 (Bennett 1969), to 85 in 1974 (Phatak 1974) to 119 in 1981 (Mandahar 1981), and the number of viruses reported to be transmitted via seeds is increasing steadily with time. In addition, viruses are also known to be readily transmitted through propagules. Infected plants may remain asymptomatic because of long latent (incubation) periods required for symptom expression in the mother plants, especially in perennial fruit trees if infection occurs late in the growing season. Planting materials prepared from such plants carry the virus(es) and symptoms of infection may be expressed when they are planted later. These plants may establish poorly, exhibit decline, and the produce from such plants may be of poor quality; infection by viruses leads to both quantitative and qualitative losses (Narayanasamy 2011). Plum pox virus (PPV) causes one of the most devastating virus diseases (sharka) infecting stone fruits in all countries. Sharka disease, once limited to Europe for most of the twentieth century, has spread to Africa, South America, North America, and Asia (OEPP/EPPO 2004). Long‐distance spread occurs primarily through grafting for producing propagation materials, and aphids may have an important role for local dispersal in some locations. Transmission of PPV via pollen also has been observed (Isac et al. 1998). The incidence of PPV reached such alarming proportions that the destruction of 648 ha of stone fruit orchards in Pennsylvania was necessary during the decade from when the disease incidence was first observed in 1999. The eradication cost was estimated at US $53 million (Welliver 2012). On the other hand, the cost associated with sharka disease management in Europe was estimated to exceed 10 billion Euros prior to 2006 (Cambra et al. 2006).

Citrus tristeza virus (CTV), existing in the form of many strains, induces different kinds of symptoms depending on the virulence of the CTV strain, cultivar, and the scion/rootstock combinations. CTV spreads primarily through infected plants and budwood; secondary spread is through different aphid species, Toxoptera citricida being most efficient in transmitting the virus from infected to healthy plants. Citrus decline due to CTV has taken a heavy toll in several countries and the virus killed more than 100 million trees propagated widely on sour orange rootstock during the last 80 years in South America, the USA, Spain, and Israel (Moreno et al. 2008). When two or more CTV strains infect citrus plants simultaneously complex symptoms appear, making it difficult to establish the identity of the strain(s). The complexity increases further because of continuous recombinations occurring in nature, resulting in the appearance of new or more virulent strains (Mukhopadhyay 2011). Severe epidemics occurred in locations where the vector T. citricida was active and was found in large populations in Brazil and Venezuela (Michaud 1998). The economic impact of CTV on the citrus industry could be lessened considerably by strengthening quarantine and certification programs and using certified virus‐tested budwood (Gergerich et al. 2015).

1.3 Nature of Seed‐ and Propagule‐Borne Microbial Pathogens

Microbial pathogens infecting plants may be carried in and/or on the seeds and propagules in an active or dormant stage. These planting materials may or may not exhibit visually recognizable symptoms of pathogen infection. Under favorable environments, the fungal pathogens may multiply and infect the emerging seedlings or become systemic and develop in the apical tissues of the plants without expressing symptoms of infection, as in the case of cereal smut diseases. Detection of the presence of the pathogens based on the symptoms alone will not be sufficient to establish the identity of the pathogens present in seeds and propagules. Several techniques – conventional methods, immunoassays, and molecular techniques – have been applied for the detection, identification, differentiation, and quantification of fungal pathogens rapidly and precisely (Chapter 2). The genetic diversity to determine the variability of isolates/strains in pathogenicity, survival ability, production of toxins, and sensitivity to environments and fungicides has to be assessed to develop effective systems of management of diseases caused by the fungal pathogens (Chapter 3). Fungal pathogens follow different paths of invasion to reach the seed tissues or storage organs. Monitoring the movement of pathogens from seed‐to‐plant and plant‐to‐seed is essential to break the vulnerable link in the life cycle of the fungal pathogens so that the progress of disease development may be arrested, resulting in the failure of infection/disease development (Chapter 4).

Bacterial pathogens with simpler structural characteristics compared to fungal