Stomatal openings on boxwood leaves yield entry portals for leaf infection by Calonectria pseudonaviculata

  • Yonghong Guo
  • James Kilcrease
  • John Hammond
  • Margaret PoolerEmail author
Short Communication


The distribution, density, and size of leaf stomata for eight boxwood taxa were investigated using both light and electron microscopy. Stomata, the pores that facilitate gaseous exchange during photosynthesis, were primarily located on the abaxial (lower) epidermis of boxwood leaves with minimal distribution on or along the central vein of the adaxial (upper) surface. Inoculation of boxwood leaves with Calonectria pseudonaviculata at different leaf sites showed that infection occurred only in areas where stomata were present. Scanning and transmission electron microscopic examination of the inoculated leaves also confirmed that the fungal pathogen gains leaf entry through stomatal openings and not by direct penetration of the cuticle. Among the eight boxwood taxa examined, stomatal densities ranged from 116 ± 15 (B. sinica ‘NaNa’) to 326 ± 21 (B. harlandii) stomata per mm2; stomatal size ranged from 500 ± 84 um2 (B. harlandii) to 1052 ± 117 um2 (B. sinica ‘NaNa’); and stomatal area per leaf (density × size) ranged from 88.63 mm2 (‘Justin Brouwers’) to 154.3 mm2 (B. harlandii). No relationship between stomatal density or size and infection rate was observed; relationships between infection rate and stomatal location and stomatal area on the abaxial surface were demonstrated.


Boxwood blight Buxus Disease resistance Electron microscopy 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Statement of human and animal rights

This article does not contain any studies with human or animal subjects performed by any of the authors.


  1. Allegre M, Daire X, Heloir M-C, Trouvelot S, Mercier L, Adrian M, Pugin A (2006) Stomatal deregulation in Plasmopara viticola- infected grapevine leaves. New Phytol 173:832–840CrossRefGoogle Scholar
  2. Daughtrey ML (2019) Boxwood blight: Threat to ornamentals. Annual Review of Phytopathology 57(1):189–209CrossRefGoogle Scholar
  3. Douglas SM (2012) Boxwood blight-A new disease for Connecticut and the U.S. The Connecticut Agricultural Experiment Station. Accessed 16 March 2018
  4. Elmhirst JF, Auxier BE (2013) First report of box blight caused by Cylindrocladium pseudonaviculata (C. buxicola) in British Columbia, Canada. Plant Dis 97:559CrossRefGoogle Scholar
  5. Gehesquiere B (2014) Cylindrocladium buxicola nom. cons. prop. (syn. Calonectria pseudonaviculata) on Buxus: molecular characterization, epidemiology, host resistance and fungicide control. Dissertation, Ghent University, BelgiumGoogle Scholar
  6. Gehesquière B, D'Haeyer S, Pham KTK, Van Kuik AJ, Maes M, Höfte M, Heungens K (2013) qPCR assays for the detection of Cylindrocladium buxicola in plant, water and air samples. Plant Dis 97:1082–1090CrossRefGoogle Scholar
  7. Guo YH, Olsen RT, Kramer M, Pooler M (2015) Effective bioassays for evaluating boxwood blight susceptibility using detached stem inoculations. HortScience 50:268–271CrossRefGoogle Scholar
  8. Guo YH, Olsen RT, Kramer M, Pooler M (2016) Use of mycelium and detached leaves in bioassays for assessing resistance to boxwood blight. Plant Dis 100:1622–1626CrossRefGoogle Scholar
  9. Hagan AK, Conner K (2013) Boxwood blight – a new disease of boxwood in the nursery and landscape in Alabama. Accessed 20 March 2018
  10. Henricot B, Culham A (2002) Cylindrocladium buxicola, a new species affecting Buxus spp., and its phylogenetic status. Mycologia 94:980–997CrossRefGoogle Scholar
  11. Henricot B, Gorton C, Denton G, Denton J (2008) Studies on the control of Cylindrocladium buxicola using fungicides and host resistance. Plant Dis 92:1273–1279CrossRefGoogle Scholar
  12. Iriarte F, Paret M, Knox G (2015) IFAS Disease alert: Boxwood blight. Accessed 16 March 2018
  13. Ivors KL, Lacey LW, Milks DC, Douglas SM, Inman MK, Marra RE, LaMondia JA (2012) First report of boxwood blight caused by Cylindrocladium pseudonaviculata in the United States. Plant Dis 96:1070CrossRefGoogle Scholar
  14. Kolattukudy PE (1985) Enzymatic penetration of the plant cuticle by fungal pathogens. Annu Rev Phytopathol 23:223–250CrossRefGoogle Scholar
  15. Kong P, Likins TM, Hong CX (2017) First report of blight of Sarcococca hookeriana var. humilis by Calonectria pseudonaviculata in Virginia. Plant Dis 101:247CrossRefGoogle Scholar
  16. Malapi-Wight M, Hebert JB, Buckley R, Daughtrey ML, Gregory NF, Rane K, Tirpak S, Crouch J (2014) First report of boxwood blight caused by Calonectria pseudonaviculata in Delaware, Maryland, New Jersey and New York. Plant Dis 98:698CrossRefGoogle Scholar
  17. Mendgen K, Hahn M, Deising H (1996) Morphogenesis and mechanisms of penetration by plant pathogenic fungi. Annu Rev Phytopathol 34:367–386CrossRefGoogle Scholar
  18. Niks RE, Rubiales D (2002) Potentially durable resistance mechanisms in plants to specialized fungal pathogens. Euphytica 124:201–216CrossRefGoogle Scholar
  19. Rubiales D, Niks RE (1996) Avoidance of rust infection by some genotypes of Hordeum chilense due to their relative inability to induce the formation of appressoria. Physio Mol Plant Pathol 49:89–101CrossRefGoogle Scholar
  20. Tucker SL, Talbot NJ (2001) Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annu Rev Phytopathol 39:385–417CrossRefGoogle Scholar
  21. Ward Gauthier NA, Beale BJ, Dixon E (2016) First report of boxwood blight caused by Calonectria pseudonaviculata in Kentucky. Plant Dis 100:1019CrossRefGoogle Scholar
  22. Williams-Woodward JL (2014) Boxwood blight found in Georgia – disease alert. Accessed 15 March 2018
  23. Wynn WK (1976) Appressorium formation over stomates by bean rust: response to a surface contact stimulus. Phytopathology 66:136–146CrossRefGoogle Scholar

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.U.S. Department of Agriculture/Agricultural Research ServiceU.S. National Arboretum, Floral and Nursery Plants Research UnitBeltsvilleUSA
  2. 2.Rutgers UniversityNew BrunswickUSA
  3. 3.Nanotechnology Systems DivHitachi High Technologies America Inc.ClarksburgUSA

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